Antigen binding polypeptides

ABSTRACT

The invention relates to a platform technology for production of antigen binding polypeptides having specificity for a desired target antigen which is based on the conventional antibody repertoire of species in the family Camelidae, and to antigen binding polypeptides obtained using this technology platform. In particular, the invention provides an antigen binding polypeptide comprising a VH domain and a VL domain, wherein at least one hypervariable loop or complementarity determining region (CDR) in the VH domain or the VL domain is obtained from a VH or VL domain of a species in the family Camelidae.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/497,239, filed Jul. 2, 2009 which claims the benefit of priority ofU.S. Provisional Application Ser. No. 61/077,730, filed Jul. 2, 2008,and U.S. Provisional Application Ser. No. 61/110,161, filed Oct. 31,2008, which is incorporated herein by reference. This application alsoclaims the benefit of foreign priority to GB 0812120.4, filed Jul. 2,2008, which is incorporated herein by reference.

FIELD OF THE INVENTION

The invention relates to a novel platform for generation of antigenbinding polypeptides, including monoclonal antibodies, which share ahigh degree of sequence and structural homology with the variabledomains of human antibodies.

BACKGROUND TO THE INVENTION

Monoclonal antibodies have many applications as research tools and,increasingly, as therapeutic or diagnostic agents. Currently more than20 different monoclonal antibodies have received regulatory approval totreat a variety of different diseases, including cancer, inflammation,auto-immune disorders, infectious disease, asthma, cardiovasculardiseases and transplant rejection and the number of monoclonal antibodydrugs in the development pipeline is increasing year-on-year.

The utility of rodent (specifically murine) monoclonal antibodies inhuman therapy is limited because of problems associated with theirnon-human origin, in particular their immunogenicity in a human host. Inorder to minimize the human immune response against therapeutic antibodydrugs, monoclonal antibody technology has evolved from full mouseantibodies to chimeric antibodies (mouse variable domains grafted on ahuman IgG backbone), to humanized antibodies (mouse CDRs grafted on ahuman IgG backbone), to “fully human” antibodies derived from syntheticlibraries or immunized transgenic mice expressing part of the human IgGrepertoire.

A number of technology platforms have been developed which allowproduction of fully human or “humanized” monoclonal antibodies againsttarget antigens of therapeutic interest. Each of these platforms has itsown particular characteristics and potential shortcomings.

Humanisation of mouse monoclonal antibodies was initially achieved bycombining mouse variable domains with human constant domains, creatingso called chimeric antibodies having about 70% of human content. Afurther degree of humanization was subsequently achieved by grafting thecomplementarity-determining regions (CDRs) of mouse monoclonalantibodies onto human framework regions of the variable antibody domainsof human antibodies. In addition, several amino acid residues present inthose framework regions were identified as interacting with the CDRs orantigen and were back mutated in the humanized antibody to improvebinding. (Almagro et al. Frontiers in Bioscience. 13: 1619-1633 (2008)).Monoclonal antibodies engineered using this approach have a relativelyhigh degree of primary sequence homology to human VH and VL domainsequences after humanisation, but a drawback is the possibility ofending up with hypervariable loops not having human-like structure,because not all mouse-encoded CDRs use canonical folds, and canonicalfold combinations, which are not found in human antibodies (Almagro etal., Mol. Immunol. 34:1199-1214 (1997); Almagro et al., Immunogen.47:355-63 (1998)). A further drawback is the large number of mutationstypically required to humanise such antibodies (the procedure for whichis complex and time-consuming), with the consequent risk of losingaffinity and potency as a result of the number of changes needed forhumanisation and, the fact that VKappa domains are mainly used in themurine repertoire, whereas approximately half of all human antibodiespossess VLambda domains.

As a potential improvement on humanised mouse monoclonal antibodies,“fully human” monoclonal antibodies can be produced by two verydifferent approaches. The first approach is selection from a fullysynthetic human combinatorial antibody library (for example HuCAL®,MorphoSys). The potential drawback of this approach is that thesynthetic library only approximates the functional diversity naturallypresent in the human germline, thus the diversity is somewhat limited.Also, antibodies generated using this approach are not derived from invivo selection of CDRs via active immunisation, and typically affinitymaturation has to be done in order to improve affinity for the targetantigen. Affinity maturation is a lengthy process which may addconsiderable time to the antibody discovery process. Also, in theprocess of affinity maturation certain amino acid residues may bechanged which may negatively affect the binding specificity or stabilityof the resulting antibody (Wu et al., J. Mol. Biol. 368: 652-65 (2007)).

Alternative “fully human” platforms are based on transgenic mice whichhave been engineered to replace the murine immunoglobulin encodingregion with antibody-encoding sequences from the human germline (forexample HuMab, Medarex). These systems have the advantage thatantibodies are raised by active immunisation, with the target antigen,i.e. they have a high starting affinity for the antigen, and that no oronly minimal antibody engineering of the original antibodies is requiredin order to make them more human-like. However, the transgenic mousestrains are by definition highly inbred and this has adverseconsequences for the strength and diversity of the antibody response.Another drawback with this platform may be impaired B cell maturationdue to human Fc/mouse Fc receptor interaction in some transgenic mousesystems.

A further platform is based on immunisation of non-human primates,specifically cynomologous monkeys. Due to the high degree of amino acidsequence identity between monkey and human immunoglobulins it ispostulated that antibodies raised in monkeys will require little or noadditional “humanisation” in the variable domains in order to renderthem useful as human therapeutics (see WO 93/02108).

SUMMARY OF THE INVENTION

The present inventors have recognised the need for a “humanised”monoclonal antibody (antigen binding polypeptide) platform which avoidssome or all of the shortcomings they have observed with prior arthumanised or fully human antibody platforms and which enables theproduction of antibodies of high specificity and affinity against abroad range of target antigens of therapeutic importance whilstminimising immunogenicity in a human host.

The present inventors have observed that both the VH and the VL domainsof conventional antibodies from the family Camelidae exhibit a highdegree of amino acid sequence identity with the VH and VL domains ofhuman antibodies over the framework regions. In fact, the degree ofsequence identity between camelid conventional VH domains and human VHdomains, and between camelid conventional VL domains and human VLdomains can approach that observed between humans and other primatespecies, e.g. cynomologous monkeys, and is much higher than might beexpected given the phylogenetic distance between humans and camelids.This finding is surprising given that the variable domains ofheavy-chain camelid antibodies (VHH) do not show this high degree ofsequence homology with human variable domains.

In addition, the inventors have observed that the hypervariable loops(H1, H2, L1, L2 and L3) of camelid VH and VL domains often exhibit ahigh degree of structural homology with the hypervariable loops of humanVH and VL domains, which is again unexpected given the evolutionarydistance between humans and camelids. The high degree of structuralhomology between camelid conventional antibodies (or rather thehypervariable loops of such antibodies) and human antibodies is alsosurprising since the hypervariable loops of heavy-chain camelidantibodies have been reported to vary substantially in conformation andlength from the corresponding loops in human and mouse VH (see review DeGenst et al., Develop Comp. Immunol. 30:187-98 (2006)).

The high degree of primary amino acid sequence homology with theframework regions of human antibodies, coupled with the high degree ofstructural homology of the antigen binding sites comprising thehypervariable loops with the binding sites of human antibodies, plus thefact that Camelidae conventional antibodies can be raised by activeimmunisation of an outbred animal population, which are phylogeneticallyquite distant from humans, has led the present inventors to surmise thatconventional antibodies from the family Camelidae are an attractivestarting point for engineering monoclonal antibodies having potentialutility as human therapeutics.

Therefore, in accordance with a first aspect of the invention there isprovided an antigen binding polypeptide comprising a VH domain and a VLdomain, wherein at least one hypervariable loop or complementaritydetermining region (CDR) in the VH domain or the VL domain is obtainedfrom a VH or VL domain of a species in the family Camelidae.

In one embodiment the antigen binding polypeptide of the invention maybe immunoreactive with a target antigen. In another embodiment theantigen binding polypeptide may bind specifically to a target antigen.

In a non-limiting embodiment the antigen binding polypeptide of theinvention may be a recombinant polypeptide.

In a non-limiting embodiment the antigen binding polypeptide of theinvention may be a chimeric polypeptide.

In a non-limiting embodiment the antigen binding polypeptide of theinvention may be a monoclonal antibody.

In a non-limiting embodiment the antigen binding polypeptide of theinvention may be a recombinantly expressed chimeric monoclonal antibody.

In a non-limiting embodiment the antigen binding polypeptide accordingto the invention may comprise hypervariable loops or complementaritydetermining regions which have been obtained by active immunisation of aspecies in the family Camelidae.

In a second aspect the invention provides a process for preparing anantigen binding polypeptide immunoreactive with a target antigen, saidprocess comprising:

-   -   (a) determining the nucleotide sequence encoding at least one        hypervariable loop or complementarity determining region (CDR)        of the VH and/or the VL domain of a Camelidae conventional        antibody immunoreactive with said target antigen; and    -   (b) expressing an antigen binding polypeptide immunoreactive        with said target antigen, said antigen binding polypeptide        comprising a VH and a VL domain, wherein at least one        hypervariable loop or complementarity determining region (CDR)        of the VH domain or the VL domain has an amino acid sequence        encoded by the nucleotide sequence determined in part (a).

In a third aspect the invention provides a process for preparing arecombinant antigen binding polypeptide that is immunoreactive with (orspecifically binds to) a target antigen, said antigen bindingpolypeptide comprising a VH domain and a VL domain, wherein at least onehypervariable loop or complementarity determining region (CDR) in the VHdomain or the VL domain is obtained from a species in the familyCamelidae, said process comprising the steps of:

-   -   (a) isolating Camelidae nucleic acid encoding at least one        hypervariable loop or complementarity determining region (CDR)        of the VH and/or the VL domain of a Camelidae conventional        antibody immunoreactive with said target antigen;    -   (b) preparing a polynucleotide comprising a nucleotide sequence        encoding hypervariable loop(s) or complementarity determining        region(s) having amino acid sequence identical to the        hypervariable loop(s) or complementarity determining region(s)        encoded by the nucleic acid isolated in step (a), which        polynucleotide encodes an antigen binding polypeptide comprising        a VH domain and a VL domain that is immunoreactive with (or        specifically binds to) said target antigen; and    -   (c) expressing said antigen binding polypeptide from the        recombinant polynucleotide of step (b).

The polynucleotide prepared in step (b) is preferably recombinant.

The invention further provides a polynucleotide comprising a nucleotidesequence which encodes an antigen binding polypeptide according to thefirst aspect of the invention, or which encodes a fragment of saidantigen binding polypeptide, which fragment comprises at least onehypervariable loop or complementarity determining region (CDR) obtainedfrom a VH or VL domain of a species in the family Camelidae.

The invention also provides an expression vector comprising thepolynucleotide defined above operably linked to regulatory sequenceswhich permit expression of the antigen binding polypeptide in a hostcell or cell-free expression system, a host cell or cell-free expressionsystem containing the expression vector, and a method of producing arecombinant antigen binding polypeptide which comprises culturing thehost cell or cell free expression system under conditions which permitexpression of the antigen binding polypeptide and recovering theexpressed antigen binding polypeptide.

Still further, the invention provides a test kit comprising an antigenbinding polypeptide according to the first aspect of the invention and apharmaceutical formulation comprising an antigen binding polypeptideaccording to the first aspect of the invention and at least onepharmaceutically acceptable diluent, excipient or carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1—shows the results of an ELISA in which sera from llamas immunisedwith IL1-Beta were tested for the presence of antibodies againstIL1-Beta, on day 0 and day 28 following immunisation.

FIG. 2—illustrates the amino acid sequences (SEQ ID NOS: 21, 255-268,respectively) of “humanized” variants of two Fabs immunoreactive withIL-1 Beta, coded 1E2 and 1F2. Based on the alignment against the closesthuman germlines, mutations in the VH and Vλ framework regions of 1E2 and1F2 were proposed. Besides the fully humanized (hum) and the wild type(wt) V regions, also a “safe variant” with only three wild type residuesremaining was proposed (safe).

FIG. 3—shows the results of an ELISA in which recombinantly expressedFabs were tested for their ability to bind biot-IL-1Beta. For this theFabs were captured on an anti-myc coated Maxisorp plate. Biotinylatedhuman IL-1Beta was added and bound cytokine was detected usingHRP-conjugated streptavidin.

FIG. 4—shows the results of phage ELISA in which phage displayinghumanized variants of Fabs 1E2 and 1F2 were tested for binding toIL-1Beta.

FIG. 5—shows the results of ELISA in which chimeric 1E2 was tested forbinding to IL-1Beta.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to a new platform technology for production ofantigen binding polypeptides having specificity for a desired targetantigen which is based on the conventional antibody repertoire ofspecies in the family Camelidae, and to antigen binding polypeptidesobtained using this technology platform.

Thus, in a first aspect the invention provides an antigen bindingpolypeptide comprising a VH domain and a VL domain, wherein at least onehypervariable loop or complementarity determining region (CDR) in the VHdomain or the VL domain is obtained from a VH or VL domain of a speciesin the family Camelidae.

In the following passages different aspects of the invention are definedin more detail. Each aspect so-defined may be combined with any otheraspect or aspects unless clearly indicated to the contrary. Inparticular, any feature indicated as being preferred or advantageous maybe combined with any other feature or features indicated as beingpreferred or advantageous.

Definitions

The term “antigen binding polypeptide” refers to any polypeptidecomprising a VH domain and a VL domain which is immunoreactive with,exhibits specific binding to, a target antigen. Exemplary antigenbinding polypeptides include antibodies and immunoglobulins, and alsoantibody fragments, as discussed elsewhere herein.

The term “antigen”, when referring to the “target antigen” against whichthe antigen binding polypeptide is immunoreactive, takes its normalmeaning to a person of ordinary skill in the art, and includes, interalia, polypeptide, peptide, polysaccharide, glycoprotein, polynucleotide(e.g. DNA), or synthetic chemical antigens.

The term “antigen” can also be used to describe the material employed inthe immunisation of animals (e.g. camelids) during the manufacture ofantigen binding polypeptides of the invention. In this context the term“antigen” may take a wider meaning, and could encompass purified formsof the antigen, and also crude or semi-purified preparations of theantigen, such as for example cells, cell lysates or supernatants, cellfractions, e.g. cell membranes, etc., plus haptens conjugated with anappropriate carrier protein. The “antigen” used in an immunisationprotocol is not necessarily structurally identical to the “targetantigen” with which the resulting antigen binding polypeptide is toimmunoreact. Typically the “antigen” used for immunisation may be atruncated form of the “target antigen”, e.g. a fragment containing animmunogenic epitope. Further characteristics of “antigens” used foractive immunisation are described elsewhere herein, and would begenerally known to a person skilled in the art.

“Specific binding” between and antigen binding polypeptide and a targetantigen refers to immunological specificity. An antigen bindingpolypeptide binds “specifically” to its target antigen if it binds anepitope on the target antigen in preference to other epitopes.

“Specific binding” does not exclude cross-reactivity with other antigensbearing similar antigenic epitopes.

“Antibodies” (Abs) and “immunoglobulins” (Igs) are glycoproteins whichexhibit binding specificity to a (target) antigen.

The camelid species are known to possess two different types ofantibodies; the classical or “conventional” antibodies and also theheavy-chain antibodies.

As used herein, the term “conventional antibody” refers to antibodies ofany isotype, including IgA, IgG, IgD, IgE or IgM. Native or naturallyoccurring “conventional” camelid antibodies are usually heterotetramericglycoproteins, composed of two identical light (L) chains and twoidentical heavy (H) chains. Each light chain is linked to a heavy chainby one covalent disulfide bond, while the number of disulfide linkagesvaries among the heavy chains of different immunoglobulin isotypes. Eachheavy and light chain also has regularly spaced intrachain disulfidebridges. Each heavy chain has at one end (N-terminal) a variable domain(VH) followed by a number of constant domains. Each light chain has avariable domain (VL) at one end (N-terminal) and a constant domain (CL)at its other end; the constant domain of the light chain is aligned withthe first constant domain of the heavy chain, and the light-chainvariable domain is aligned with the variable domain of the heavy chain.Particular amino acid residues are believed to form an interface betweenthe light- and heavy-chain variable domains.

The term “heavy-chain antibody” refers to the second type of antibodiesknown to occur naturally in camelid species, such antibodies beingnaturally devoid of light chains (Hamers-Casterman, et al. Nature. 1993;363; 446-8). The heavy-chain antibodies (abbreviated to HCAb) arecomposed of two heavy chains linked by a covalent disulphide bond. Eachheavy chain in the HCAb has a variable domain at one end. The variabledomains of HCAbs are referred to as “VHH” in order to distinguish themfrom the variable domains of the heavy chains of “conventional” camelidantibodies (VH). The VHH domains and VH domains are entirely distinctand are encoded by different gene segments in the camelid genome.

The VL domains in the polypeptide of the invention may be of the VLambdatype or the Vkappa type. The term “VL domain” therefore refers to bothVKappa and VLambda isotypes from Camelidae, and engineered variantsthereof which contain one or more amino acid substitutions, insertionsor deletions relative to a Camelidae VL domain.

The term “VH domain” refers to a VH domain of any known heavy chainisotype of Camelidae, including γ, ε, δ, α or β isotypes, as well asengineered variants thereof which contain one or more amino acidsubstitutions, insertions or deletions relative to a Camelidae VHdomain. The term “VH domain” refers only to VH domains of camelidconventional antibodies and does not encompass camelid VHH domains.

The term “variable” refers to the fact that certain portions of thevariable domains VH and VL differ extensively in sequence amongantibodies and are used in the binding and specificity of eachparticular antibody for its target antigen. However, the variability isnot evenly distributed throughout the variable domains of antibodies. Itis concentrated in three segments called “hypervariable loops” in eachof the VL domain and the VH domain which form part of the antigenbinding site. The first, second and third hypervariable loops of theVLambda light chain domain are referred to herein as L1(λ), L2(λ) andL3(λ) and may be defined as comprising residues 24-33 (L1(λ), consistingof 9, 10 or 11 amino acid residues), 49-53 (L2(λ), consisting of 3residues) and 90-96 (L3(λ), consisting of 5 residues) in the VL domain(Morea et al., Methods 20:267-279 (2000)). The first, second and thirdhypervariable loops of the VKappa light chain domain are referred toherein as L1(κ), L2(κ) and L3(κ) and may be defined as comprisingresidues 25-33 (L1(κ), consisting of 6, 7, 8, 11, 12 or 13 residues),49-53 (L2(κ), consisting of 3 residues) and 90-97 (L3(κ), consisting of6 residues) in the VL domain (Morea et al., Methods 20:267-279 (2000)).The first, second and third hypervariable loops of the VH domain arereferred to herein as H1, H2 and H3 and may be defined as comprisingresidues 25-33 (H1, consisting of 7, 8 or 9 residues), 52-56 (H2,consisting of 3 or 4 residues) and 91-105 (H3, highly variable inlength) in the VH domain (Morea et al., Methods 20:267-279 (2000)).

Unless otherwise indicated, the terms L1, L2 and L3 respectively referto the first, second and third hypervariable loops of a VL domain, andencompass hypervariable loops obtained from both Vkappa and Vlambdaisotypes from Camelidae. The terms H1, H2 and H3 respectively refer tothe first, second and third hypervariable loops of the VH domain, andencompass hypervariable loops obtained from any of the known heavy chainisotypes from Camelidae, including γ, ε, δ, α or μ.

The hypervariable loops L1, L2, L3, H1, H2 and H3 may each comprise partof a “complementarity determining region” or “CDR”. The terms“hypervariable loop” and “complementarity determining region” are notstrictly synonymous, since the hypervariable loops (HVs) are defined onthe basis of structure, whereas complementarity determining regions(CDRs) are defined based on sequence variability (Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md., 1983) and thelimits of the HVs and the CDRs may be different in some VH and VLdomains.

The CDRs of the VL and VH domains can typically be defined as comprisingthe following amino acids: residues 24-34 (CDRL1), 50-56 (CDRL2) and89-97 (CDRL3) in the light chain variable domain, and residues 31-35 or31-35b (CDRH1), 50-65 (CDRH2) and 95-102 (CDRH3) in the heavy chainvariable domain; Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md. (1991)). Thus, the HVs may be comprised within thecorresponding CDRs and references herein to the “hypervariable loops” ofVH and VL domains should be interpreted as also encompassing thecorresponding CDRs, and vice versa, unless otherwise indicated.

The more highly conserved portions of variable domains are called theframework region (FR). The variable domains of native heavy and lightchains each comprise four FRs (FR1, FR2, FR3 and FR4, respectively),largely adopting a β-sheet configuration, connected by the threehypervariable loops. The hypervariable loops in each chain are heldtogether in close proximity by the FRs and, with the hypervariable loopsfrom the other chain, contribute to the formation of the antigen-bindingsite of antibodies. Structural analysis of antibodies revealed therelationship between the sequence and the shape of the binding siteformed by the complementarity determining regions (Chothia et al., J.Mol. Biol. 227: 799-817 (1992)); Tramontano et al., J. Mol. Biol,215:175-182 (1990)). Despite their high sequence variability, five ofthe six loops adopt just a small repertoire of main-chain conformations,called “canonical structures”. These conformations are first of alldetermined by the length of the loops and secondly by the presence ofkey residues at certain positions in the loops and in the frameworkregions that determine the conformation through their packing, hydrogenbonding or the ability to assume unusual main-chain conformations.

The constant domains are not involved directly in binding of an antibodyto an antigen, but exhibit various effector functions, such asparticipation of the antibody in antibody-dependent cellular toxicity(ADCC) or complement-dependent cytotoxicity (CDC).

In all aspects and embodiments of the invention, the Camelidae (orcamelid) species (from which the hypervariable loops or CDRs of theantigen binding polypeptide of the invention are obtained) can be camel,llama, dromedary, vicunia, guanaco or alpaca and any crossings thereof.Llama (Lama glama) and alpaca (Lama pacos) are the preferred Camelidaespecies for all aspects of the invention.

The antigen binding polypeptides of the invention are characterised inthat they contain at least one hypervariable loop or complementaritydetermining region which is obtained from a VH domain or a VL domain ofa species in the family Camelidae. For the avoidance of doubt, the terms“VH domain” “VL domain” refer to domains derived from camelidconventional antibodies. This definition excludes the camelid heavychain only VHH antibodies, and recombinant constructs containing solelyHVs or CDRs of camelid VHH domains, which are not encompassed within thescope of the present invention.

By “hypervariable loop or complementarity determining region obtainedfrom a VH domain or a VL domain of a species in the family Camelidae” ismeant that that hypervariable loop (HV) or CDR has an amino acidsequence which is identical, or substantially identical, to the aminoacid sequence of a hypervariable loop or CDR which is encoded by aCamelidae immunoglobulin gene. In this context “immunoglobulin gene”includes germline genes, immunoglobulin genes which have undergonerearrangement, and also somatically mutated genes. Thus, the amino acidsequence of the HV or CDR obtained from a VH or VL domain of a Camelidaespecies may be identical to the amino acid sequence of a HV or CDRpresent in a mature Camelidae conventional antibody. The term “obtainedfrom” in this context implies a structural relationship, in the sensethat the HVs or CDRs of the antigen binding polypeptide of the inventionembody an amino acid sequence (or minor variants thereof) which wasoriginally encoded by a Camelidae immunoglobulin gene. However, thisdoes not necessarily imply a particular relationship in terms of theproduction process used to prepare the antigen binding polypeptide ofthe invention. As will be discussed below, there are several processeswhich may be used to prepare antigen binding polypeptides comprising HVsor CDRs with amino acid sequences identical to (or substantiallyidentical to) sequences originally encoded by a Camelidae immunoglobulingene.

For the avoidance of doubt, the terms “VH domain of a conventionalantibody of a camelid” and “VH domain obtained from a species ofCamelidae” are used synonymously and encompass VH domains which are theproducts of synthetic or engineered recombinant genes (includingcodon-optimised synthetic genes), which VH domains have an amino acidsequence identical to (or substantially identical to) the amino acidsequence of a VH domain encoded by a Camelidae immunoglobulin gene(germline, rearranged or somatically mutated). Similarly, the terms “VLdomain of a conventional antibody of a camelid” and “VL domain obtainedfrom a species of Camelidae” are used synonymously and encompass VLdomains which are the products of synthetic or engineered recombinantgenes (including codon-optimised synthetic genes), which VL domains havean amino acid sequence identical to (or substantially identical to) theamino acid sequence of a VL domain encoded by a Camelidae immunoglobulingene (germline, rearranged or somatically mutated).

The antigen binding polypeptides of the invention are typicallyrecombinantly expressed polypeptides, and may be chimeric polypeptides.The term “chimeric polypeptide” refers to an artificial (non-naturallyoccurring) polypeptide which is created by juxtaposition of two or morepeptide fragments which do not otherwise occur contiguously. Includedwithin this definition are “species” chimeric polypeptides created byjuxtaposition of peptide fragments encoded by two or more species, e.g.camelid and human.

The antigen binding polypeptides of the invention are not naturallyoccurring human antibodies, specifically human autoantibodies, due tothe requirement for at least one hypervariable loop (or CDR) fromcamelid. By “naturally occurring” human antibody is meant an antibodywhich is naturally expressed within a human subject. Antigen bindingpolypeptides having an amino acid sequence which is 100% identical tothe amino acid sequence of a naturally occurring human antibody, or afragment thereof, which natural antibody or fragment is not chimeric andhas not been subject to any engineered changes in amino acid sequence(excluding somatic mutations) are excluded from the scope of theinvention.

The antigen binding polypeptides according to the invention compriseboth a heavy chain variable (VH) domain and a light chain variable (VL)domain, and are characterised in that at least one hypervariable loop orcomplementarity determining region in either the VH domain or the VLdomain is obtained from a species in the family Camelidae.

In alternative embodiments, either H1 or H2, or both H1 and H2 in the VHdomain may be obtained from a species in the family Camelidae, andindependently either L1 or L2 or both L1 and L2 in the VL domain may beobtained from a species in the family Camelidae. In further embodimentsH3 in the VH domain or L3 in the VL domain may also be obtained from aspecies in the family Camelidae. All possible permutations of theforegoing are permitted.

In one specific embodiment each of the hypervariable loops H1, H2, H3,L1, L2 and L3 in both the VH domain and the VL domain may be obtainedfrom a species in the family Camelidae.

In one embodiment the entire VH domain and/or the entire VL domain maybe obtained from a species in the family Camelidae. The Camelidae VHdomain and/or the Camelidae VL domain may then be subject to proteinengineering, in which one or more amino acid substitutions, insertionsor deletions are introduced into the Camelidae sequence. Theseengineered changes preferably include amino acid substitutions relativeto the Camelidae sequence. Such changes include “humanisation” or“germlining” wherein one or more amino acid residues in acamelid-encoded VH or VL domain are replaced with equivalent residuesfrom a homologous human-encoded VH or VL domain.

In certain embodiments, Camelidae hypervariable loops (or CDRs) may beobtained by active immunisation of a species in the family Camelidaewith a desired target antigen. As discussed and exemplified in detailherein, following immunisation of Camelidae (either the native animal ora transgenic animal, engineered to express the immunoglobulin repertoireof a camelid species) with the target antigen, B cells producing(conventional Camelidae) antibodies having specificity for the desiredantigen can be identified and polynucleotide encoding the VH and VLdomains of such antibodies can be isolated using known techniques.

Thus, in a specific embodiment, the invention provides a recombinantantigen binding polypeptide immunoreactive with a target antigen, thepolypeptide comprising a VH domain and a VL domain, wherein at least onehypervariable loop or complementarity determining region in the VHdomain or the VL domain is obtained from a VH or VL domain of a speciesin the family Camelidae, which antigen binding polypeptide is obtainableby a process comprising the steps of:

-   -   (a) immunising a species in the family Camelidae with a target        antigen or with a polynucleotide encoding said target antigen        and raising an antibody to said target antigen;    -   (b) determining the nucleotide sequence encoding at least one        hypervariable loop or complementarity determining region (CDR)        of the VH and/or the VL domain of a Camelidae conventional        antibody immunoreactive with said target antigen; and    -   (c) expressing an antigen binding polypeptide immunoreactive        with said target antigen, said antigen binding polypeptide        comprising a VH and a VL domain, wherein at least one        hypervariable loop or complementarity determining region (CDR)        of the VH domain or the VL domain has an amino acid sequence        encoded by the nucleotide sequence determined in part (a).

Isolated Camelidae VH and VL domains obtained by active immunisation canbe used as a basis for engineering antigen binding polypeptidesaccording to the invention. Starting from intact Camelidae VH and VLdomains, it is possible to engineer one or more amino acidsubstitutions, insertions or deletions which depart from the startingCamelidae sequence. In certain embodiments, such substitutions,insertions or deletions may be present in the framework regions of theVH domain and/or the VL domain. The purpose of such changes in primaryamino acid sequence may be to reduce presumably unfavourable properties(e.g. immunogenicity in a human host (so-called humanization), sites ofpotential product heterogeneity and or instability (glycosylation,deamidation, isomerisation, etc.) or to enhance some other favourableproperty of the molecule (e.g. solubility, stability, bioavailability,etc.). In other embodiments, changes in primary amino acid sequence canbe engineered in one or more of the hypervariable loops (or CDRs) of aCamelidae VH and/or VL domain obtained by active immunisation. Suchchanges may be introduced in order to enhance antigen binding affinityand/or specificity, or to reduce presumably unfavourable properties,e.g. immunogenicity in a human host (so-called humanization), sites ofpotential product heterogeneity and or instability, glycosylation,deamidation, isomerisation, etc., or, to enhance some other favourableproperty of the molecule, e.g. solubility, stability, bioavailability,etc.

Thus, in one embodiment, the invention provides a recombinant antigenbinding polypeptide which contains at least one amino acid substitutionin at least one framework or CDR region of either the VH domain or theVL domain in comparison to a Camelidae VH or VL domain obtained byactive immunisation of a species in the family Camelidae with a targetantigen. This particular embodiment excludes antigen bindingpolypeptides containing native Camelidae VH and VL domains produced byactive immunisation

As an alternative to “active immunisation” with a target antigen (or acomposition comprising the target antigen or a polynucleotide encodingit) it is also possible to make use of immune responses in diseasedCamelidae animals or naturally occurring immune responses withinCamelidae species as a source of VH and/or VL domains which can be usedas components of antigen binding polypeptides with the desiredantigen-binding properties. Such VH/VL domains may also be used as thestarting point for engineering antigen-binding polypeptides in ananalogous manner to VH/VL domains obtained by active immunisation. Theinvention still further encompasses the use of non-immune libraries, andto antigen-binding polypeptides obtained/derived therefrom.

In other embodiments, the invention encompasses “chimeric” antibodymolecules comprising VH and VL domains from Camelidae (or engineeredvariants thereof) and one or more constant domains from a non-camelidantibody, for example human-encoded constant domains (or engineeredvariants thereof). The invention also extends to chimeric antigenbinding polypeptides (e.g. antibody molecules) wherein one of the VH orthe VL domain is camelid-encoded, and the other variable domain isnon-camelid (e.g. human). In such embodiments it is preferred that boththe VH domain and the VL domain are obtained from the same species ofcamelid, for example both VH and VL may be from Lama glama or both VHand VL may be from Lama pacos (prior to introduction of engineered aminoacid sequence variation). In such embodiments both the VH and the VLdomain may be derived from a single animal, particularly a single animalwhich has been actively immunised.

As an alternative to engineering changes in the primary amino acidsequence of Camelidae VH and/or VL domains, individual Camelidaehypervariable loops or CDRs, or combinations thereof, can be isolatedfrom Camelidae VH/VL domains and transferred to an alternative (i.e.non-Camelidae) framework, e.g. a human VH/VL framework, by CDR grafting.

Sequence Identity/Homology with Human Variable Domains

The present inventors have observed that Camelidae germline andsomatically mutated DNA sequences encoding both the VH and the VLdomains of conventional antibodies from species in the family Camelidaeexhibit a high degree of sequence identity/sequence homology with thehuman germline DNA sequences which encode VH and VL domains of humanantibodies, over the framework regions.

Thus, the antigen binding polypeptides of the invention arecharacterised in that they exhibit a high degree of amino acid sequencehomology with VH and VL domains of human antibodies.

In one embodiment the VH domain of the antigen binding polypeptideaccording to the invention may exhibit an amino acid sequence identityor sequence homology of 80% or greater with one or more human VH domainsacross the framework regions FR1, FR2, FR3 and FR4. In other embodimentsthe amino acid sequence identity or sequence homology between the VHdomain of the polypeptide of the invention and one or more human VHdomains may be 85% or greater, 90% or greater, 95% or greater, 97% orgreater, or up to 99% or even 100%, of course with the proviso that atleast one hypervariable loop or CDR is obtained from Camelidae, i.e. hasan amino acid sequence which is identical (or substantially identical)to the amino acid sequence of a hypervariable loop or CDR encoded by aCamelidae VH or VL gene.

In one embodiment the VH domain of the polypeptide of the invention maycontain one or more amino acid sequence mis-matches across the frameworkregions FR1, FR2, FR3 and FR4, in comparison to the closest matchedhuman VH sequence. This latter embodiment would expressly excludepolypeptides comprising a VH domain, or both VH and VL domains, in whichthe framework region has entirely human sequence.

In another embodiment the VL domain of the antigen binding polypeptideaccording to the invention may exhibit a sequence identity or sequencehomology of 80% or greater with one or more human VL domains across theframework regions FR1, FR2, FR3 and FR4. In other embodiments the aminoacid sequence identity or sequence homology between the VL domain of thepolypeptide of the invention and one or more human VL domains may be 80%or greater 90% or greater, 95% or greater, 97% or greater, or up to 99%or even 100%.

In one embodiment the VL domain of the polypeptide of the invention maycontain one or more amino acid sequence mis-matches across the frameworkregions FR1, FR2, FR3 and FR4, in comparison to the closest matchedhuman VL sequence. This latter embodiment would expressly excludepolypeptides comprising VL domain, or both VL and VH domains in whichthe framework region has entirely human sequence.

The antigen binding polypeptide of the invention may comprise a “fullyhuman” VH or VL domain, provided that only one fully human variabledomain is present, and then in combination with a variable domaincomprising hypervariable loop(s) or CDR(s) obtained from Camelidae.

Representative alignments of Camelidae and human germline sequencesincluded in the accompanying examples reveal that the conventionalcamelid VH and VL domains exhibit a remarkably high sequence homology totheir human counterparts. From these examples it can be concluded thattypically less than 8, and often only as few as 5 amino acid residuespresent in the framework regions of a VH or VL domain differ in a givenposition from the closest human germline-encoded sequences. Given thatthere are no structural limitations associated with those positions,humanization by site directed mutagenesis is expected to bestraightforward.

Therefore, in a particular embodiment, the antigen binding polypeptidesof the invention may comprise VH and/or VL domains of conventionalCamelidae antibodies, for example conventional Camelidae antibodiesobtained (obtainable) by active immunisation of camelidae with a targetantigen (or polynucleotide encoding the target antigen), wherein said VHand VL domains have been (independently) engineered to introduce a totalof between 1 and 10, i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acidsubstitutions across the framework regions FR1, FR2, FR3 and FR4 ineither one or both of the VH domain and the VL domain. Such amino acidsubstitutions may include (but are not limited to) substitutions whichresult in “humanisation”, by replacing mis-matched amino acid residuesin a starting Camelidae VH or VL domain with the equivalent residuefound in a human germline-encoded VH or VL domain. It is also possibleto independently make amino acid substitutions in the hypervariableloops (CDRs) of said camelid-derived VH and VL domains, and suchvariants may form part of the present invention. References herein to“amino acid substitutions” include substitutions in which a naturallyoccurring amino acid is replaced with a non-natural amino acid, or anamino acid subjected to post-translational modification.

Before analyzing the percentage sequence identity between Camelidae andhuman germline VH and VL, the canonical folds may be determined, whichallows the identification of the family of human germline segments withthe identical combination of canonical folds for H1 and H2 or L1 and L2(and L3). Subsequently the human germline family member that has thehighest degree of sequence homology with the Camelidae variable regionof interest is chosen for scoring the sequence homology. Thedetermination of Chothia canonical classes of hypervariable loops L1,L2, L3, H1 and H2 was performed with the bioinformatics tools publiclyavailable on webpage www.bioinf.org.uk/abs/chothia.html.page. The outputof the program shows the key residue requirements in a datafile. Inthese datafiles, the key residue positions are shown with the allowedamino acids at each position. The sequence of the variable region of theantibody is given as input and is first aligned with a consensusantibody sequence to assign the Kabat numbering scheme. The analysis ofthe canonical folds uses a set of key residue templates derived by anautomated method developed by Martin and Thornton (Martin et al., J.Mol. Biol. 263:800-815 (1996)).

With the particular human germline V segment known, which uses the samecombination of canonical folds for H1 and H2 or L1 and L2 (and L3), thebest matching family member in terms of sequence homology was determinedWith bioinformatics tools the percentage sequence identity betweenCamelidae VH and VL domain framework amino acid sequences andcorresponding sequences encoded by the human germline can be determined,but actually manual aligning of the sequences can be applied as well.Human immunoglobulin sequences can be identified from several proteindata bases, such as VBase (vbase.mrc-cpe.cam.ac.uk) or thePluckthun/Honegger database (www.bioc.unizhch/antibody/Sequences/Germlines). To compare the human sequences to theV regions of Camelidae VH or VL domains a sequence alignment algorithmsuch as available via websites like www.expasy.ch/toolsflialign can beused, but also manual alignment with the limited set of sequences can beperformed. Human germline light and heavy chain sequences of thefamilies with the same combinations of canonical folds and with thehighest degree of homology with the framework regions 1, 2, and 3 ofeach chain are selected and compared with the Camelidae variable regionof interest; also the FR4 is checked against the human germline JH andJK or JL regions.

Note that in the calculation of overall percent sequence homology theresidues of FR1, FR2 and FR3 are evaluated using the closest matchsequence from the human germline family with the identical combinationof canonical folds. Only residues different from the closest match orother members of the same family with the same combination of canonicalfolds are scored (NB—excluding any primer-encoded differences). However,for the purposes of humanization, residues in framework regionsidentical to members of other human germline families, which do not havethe same combination of canonical folds, can be considered “human”,despite the fact that these are scored “negative” according to thestringent conditions described above. This assumption is based on the“mix and match” approach for humanization, in which each of FR1, FR2,FR3 and FR4 is separately compared to its closest matching humangermline sequence and the humanized molecule therefore contains acombination of different FRs as was done by Qu and colleagues (Qu etal., Clin. Cancer Res. 5:3095-3100 (1999)) and Ono and colleagues (Onoet al., Mol. Immunol. 36:387-395 (1999)).

The boundaries of the individual framework regions may be assigned usingthe IMGT numbering scheme, which is an adaptation of the numberingscheme of Chothia (Lefranc et al., NAR 27: 209-212 (1999);imgt.cines.fr).

Despite the unexpectedly high sequence homology between Camelidae andhuman across the framework regions of the VH and VL domains, it isnevertheless possible to distinguish camelid-encoded hypervariable loops(CDRs) from human-encoded hypervariable loops (CDRs) by straightforwardsequence comparison with camelid and human germline VH and VL sequences.

Structural Homology with Human-Encoded VH and VL Domains

A preferred embodiment is also to use Camelid hypervariable loops orCDRs having human or human-like canonical folds, as discussed in detailbelow.

Thus, in one embodiment at least one hypervariable loop or CDR in eitherthe VH domain or the VL domain of the antigen binding polypeptide of theinvention is obtained from a VH or VL domain obtained from a species ofCamelidae, yet exhibits a predicted or actual canonical fold structurewhich is substantially identical to a canonical fold structure whichoccurs in human antibodies.

It is well established in the art that although the primary amino acidsequences of hypervariable loops present in both VH domains and VLdomains encoded by the human germline are, by definition, highlyvariable, all hypervariable loops, except CDR H3 of the VH domain, adoptonly a few distinct structural conformations, termed canonical folds(Chothia et al., J. Mol. Biol. 196:901-917 (1987); Tramontano et al.Proteins 6:382-94 (1989)), which depend on both the length of thehypervariable loop and presence of the so-called canonical amino acidresidues (Chothia et al., J. Mol. Biol. 196:901-917 (1987)). Actualcanonical structures of the hypervariable loops in intact VH or VLdomains can be determined by structural analysis (e.g. X-raycrystallography), but it is also possible to predict canonical structureon the basis of key amino acid residues which are characteristic of aparticular structure (discussed further below). In essence, the specificpattern of residues that determines each canonical structure forms a“signature” which enables the canonical structure to be recognised inhypervariable loops of a VH or VL domain of unknown structure; canonicalstructures can therefore be predicted on the basis of primary amino acidsequence alone.

Based on analysis of germline and somatically mutated VH and VLsequences, the present inventors predict that the hypervariable loops ofCamelidae VH and VL domains (with the exception of H3 in the VH domainand sometimes also L3 in the VL domain) also adopt canonical foldstructures which are substantially identical to canonical foldstructures adopted by the hypervariable loops of human antibodies.

The predicted canonical fold structures for the hypervariable loops ofany given VH or VL sequence in an antigen binding polypeptide can beanalysed using algorithms which are publicly available fromwww.bioinf.org.uk/abs/chothia.html,www.biochem.ucl.ac.uk/˜martin/antibodies.html andwww.bioc.unizh.ch/antibody/Sequences/GermlinesVbase_hVk.html. Thesetools permit query VH or VL sequences to be aligned against human VH orVL domain sequences of known canonical structure, and a prediction ofcanonical structure made for the hypervariable loops of the querysequence.

In the case of the VH domain, H1 and H2 loops derived from Camelidae maybe scored as having a canonical fold structure “substantially identical”to a canonical fold structure known to occur in human antibodies if atleast the first, and preferable both, of the following criteria arefulfilled:

1. An identical length, determined by the number of residues, to theclosest matching human canonical structural class.

2. At least 33% identity, preferably at least 50% identity with the keyamino acid residues described for the corresponding human H1 and H2canonical structural classes.

(note for the purposes of the foregoing analysis the H1 and H2 loops aretreated separately and each compared against its closest matching humancanonical structural class)

The foregoing analysis relies on prediction of the canonical structureof the Camelidae H1 and H2 loops. If the actual structure of the H1 andH2 loops is known, for example based on X-ray crystallography, then theH1 and H2 loops derived from Camelidae may also be scored as having acanonical fold structure “substantially identical” to a canonical foldstructure known to occur in human antibodies if the length of the loopdiffers from that of the closest matching human canonical structuralclass (typically by ±1 or ±2 amino acids) but the actual structure ofthe Camelidae H1 and H2 loops matches the structure of a human canonicalfold.

Key amino acid residues found in the human canonical structural classesfor the first and second hypervariable loops of human VH domains (H1 andH2) are described by Chothia et al., J. Mol. Biol. 227:799-817 (1992),the contents of which are incorporated herein in their entirety byreference. In particular, Table 3 on page 802 of Chothia et al., whichis specifically incorporated herein by reference, lists preferred aminoacid residues at key sites for H1 canonical structures found in thehuman germline, whereas Table 4 on page 803, also specificallyincorporated by reference, lists preferred amino acid residues at keysites for CDR H2 canonical structures found in the human germline.

The accompanying examples contain an analysis of germline VH sequencesfrom Camelidae species (specifically llama and dromedary) comparing theactual amino acid residues found in Camelidae versus the amino acidresidues in the closest human germline VH sequence, for each of thepositions in H1 and H2, and underlying framework regions, considered tobe key for the canonical fold structure according to the criteria ofChothia et al., J Mol Biol. 227:799-817 (1992). It is observed that thenumber of identical key residues between camelid and human is most oftenabove 33%, and typically in the range of from 50 to 100%.

In one embodiment, both H1 and H2 in the VH domain of the antigenbinding polypeptide of the invention are obtained from a VH domain of aCamelidae species, yet exhibit a predicted or actual canonical foldstructure which is substantially identical to a canonical fold structurewhich occurs in human antibodies.

The inventors surmise that it is important not only for thehypervariable loops, specifically H1 and H2 in the VH domain,individually to have canonical structures of a type which occursnaturally in human antibodies, it is also important for H1 and H2 in anygiven VH domain to form a combination of canonical fold structures whichis identical to a combination of canonical structures known to occur inat least one human germline VH domain. It has been observed that onlycertain combinations of canonical fold structures at H1 and H2 actuallyoccur in VH domains encoded by the human germline. The present inventorswere surprised to discover that every available Camelidae germline orsomatically mutated VH sequence which could be analysed exhibited notonly individual canonical fold structures at H1 and H2 substantiallyidentical to those used in human antibodies, but also the correctcombinations of structures at H1 and H2 to match combinations found inhuman antibodies. This represents a distinct advantage over otherplatforms for production of antibodies for potential therapeutic use inhumans which may produce antibodies having “correct” human-likecanonical fold structures at H1 and H2 but in a combination which doesnot occur in human antibodies. By way of example, the inventors' ownanalysis of the structure of antibodies derived from non-human primates(Biogen IDEC's galiximab (anti-CD80) an lumiliximab (anti-CD23) and thenon-human primate mAb against Anthrax Toxin from Pelat et al., J. Mol.Biol. 384:1400-7 (2008)) indicates that structurally they are notconsistently very close to the human antibody structure, particularlyhaving regard to the combination of canonical folds. The absence of acorrect combination of canonical folds at H1 and H2 could lead to agiven antigen binding polypeptide (which is “humanised” in the frameworkregions) being immunogenic in a human host.

Thus, in a further embodiment H1 and H2 in the VH domain of the antigenbinding polypeptide of the invention are obtained from a VH domain of aCamelidae species, yet form a combination of predicted or actualcanonical fold structures which is identical to a combination ofcanonical fold structures known to occur in a human germline orsomatically mutated VH domain.

In non-limiting embodiments H1 and H2 in the VH domain of the antigenbinding polypeptide of the invention are obtained from a VH domain of aCamelidae species, and form one of the following canonical foldcombinations: 1-1, 1-2, 1-3, 1-6, 1-4, 2-1, 3-1 and 3-5.

It is preferred that the VH domain of the antigen binding polypeptide ofthe invention exhibit both high sequence identity/sequence homology withhuman VH, and also that the hypervariable loops in the VH domain exhibitstructural homology with human VH.

It may be advantageous for the canonical folds present at H1 and H2 inthe VH domain of the antigen binding polypeptide according to theinvention, and the combination thereof, to be “correct” for the human VHgermline sequence which represents the closest match with the VH domainof the antigen binding polypeptide of the invention in terms of overallprimary amino acid sequence identity. By way of example, if the closestsequence match is with a human germline VH3 domain, then it may beadvantageous for H1 and H2 (obtained from Camelidae) to form acombination of canonical folds which also occurs naturally in a humanVH3 domain.

Thus, in one embodiment the VH domain of the antigen binding polypeptideof the invention may exhibit a sequence identity or sequence homology of80% or greater, 85% or greater, 90% or greater, 95% or greater, 97% orgreater, or up to 99% or even 100% with a human VH domain across theframework regions FR1, FR2, FR3 and FR4, and in addition H1 and H2 inthe same antigen binding polypeptide are obtained from a VH domain of aCamelidae species, but form a combination of predicted or actualcanonical fold structures which is the same as a canonical foldcombination known to occur naturally in the same human VH domain.

In other embodiments, L1 and L2 in the VL domain of the antigen bindingpolypeptide of the invention are each obtained from a VL domain of aCamelidae species, and each exhibit a predicted or actual canonical foldstructure which is substantially identical to a canonical fold structurewhich occurs in human antibodies.

As with the VH domains, the hypervariable loops of VL domains of bothVLambda and VKappa types can adopt a limited number of conformations orcanonical structures, determined in part by length and also by thepresence of key amino acid residues at certain canonical positions.

L1, L2 and L3 loops obtained from a VL domain of a Camelidae species,yet may be scored as having a canonical fold structure “substantiallyidentical” to a canonical fold structure known to occur in humanantibodies if at least the first, and preferable both, of the followingcriteria are fulfilled:

1. An identical length, determined by the number of residues, to theclosest matching human structural class.

2. At least 33% identity, preferably at least 50% identity with the keyamino acid residues described for the corresponding human L1 or L2canonical structural classes, from either the VLambda or the VKapparepertoire.

(note for the purposes of the foregoing analysis the L1 and L2 loops aretreated separately and each compared against its closest matching humancanonical structural class)

The foregoing analysis relies on prediction of the canonical structureof the Camelidae L1, L2 and L3 loops. If the actual structure of the L1,L2 and L3 loops is known, for example based on X-ray crystallography,then L1, L2 or L3 loops derived from Camelidae may also be scored ashaving a canonical fold structure “substantially identical” to acanonical fold structure known to occur in human antibodies if thelength of the loop differs from that of the closest matching humancanonical structural class (typically by ±1 or ±2 amino acids) but theactual structure of the Camelidae loops matches a human canonical fold.

Key amino acid residues found in the human canonical structural classesfor the CDRs of human VLambda and VKappa domains are described by Moreaet al. Methods, 20: 267-279 (2000) and Martin et al., J. Mol. Biol.,263:800-815 (1996). The structural repertoire of the human VKappa domainis also described by Tomlinson et al. EMBO J. 14:4628-4638 (1995), andthat of the VLambda domain by Williams et al. J. Mol. Biol., 264:220-232(1996). The contents of all these documents are to be incorporatedherein by reference.

The accompanying examples contain an analysis of germline VL sequencesor both kappa and lambda type from Camelidae species (specifically llamaand dromedary), comparing the actual amino acid residues found inCamelidae versus the amino acid residues in the closest human germlineVLambda or VKappa sequence, for each of the positions in L1 and L2considered to be key for the canonical fold structure. It is observedthat the number of identical key residues between camelid and human istypically in the range of from 33 to 100%, more often between 50 to100%, typically closer to 100%.

L1 and L2 in the VL domain may form a combination of predicted or actualcanonical fold structures which is identical to a combination ofcanonical fold structures known to occur in a human germline VL domain.

In non-limiting embodiments L1 and L2 in the VLambda domain may form oneof the following canonical fold combinations: 11-7, 13-7(A,B,C),14-7(A,B), 12-11, 14-11 and 12-12 (as defined in Williams et al. J. Mol.Biol. 264:220-32 (1996) and as shown onwww.bioc.uzh.ch/antibody/Sequences/Germlines/VBase_hVL). In non-limitingembodiments L1 and L2 in the Vkappa domain may form one of the followingcanonical fold combinations: 2-1, 3-1, 4-1 and 6-1 (as defined inTomlinson et al. EMBO J. 14:4628-38 (1995) and as shown onwww.bioc.uzh.ch/antibody/Sequences/Germlines/VBase_hVK

In a further embodiment, all three of L1, L2 and L3 in the VL domain mayexhibit a substantially human structure. Most human Vκ germline segmentsencode also a single conformation of the L3 loop (type 1), which isstabilized by the conserved cis-proline on position 95, but otherconformations in rearranged sequences are possible due to the process ofV-J joining and the potential loss of this proline residue. The publiclyavailable somatically mutated dromedary Vκ sequences have a type 1canonical fold for L3(x) like is found in human kappa germlinesequences, and Proline on position 95 occurs in six out of sevendromedary Vκ domains. Therefore, where the antigen binding polypeptidecontains a Vκ domain, this domain may possess the conserved Prolineresidue on position 95.

The structural repertoire of the human VL germline sequences wasanalyzed by Williams and colleagues (Williams et al., J. Mol. Biol.264:220-232 (1996)). The three families analyzed here encode identicalconformations of the L2 loop. The L3 loop conformation is thought to bemore highly variable, as there is some length variation and nocis-proline residue. Indeed the available somatically mutated dromedaryVλ sequences show a high variability in the length of L3. Most of thesehave a canonical fold for L3 (f.i. VLambda 3-1 family members Camvl19(10A) and Camvl20 (1/9A), VLambda 2-18 family members Camvl5, 17, 30, 36and 52 (all 10B) and VLambda 1-40 family member Camvl44 (5/11A)).

It is preferred that the VL domain of the antigen binding polypeptide ofthe invention exhibit both high sequence identity/sequence homology withhuman VL, and also that the hypervariable loops in the VL domain exhibitstructural homology with human VL.

In one embodiment, the VL domain of the antigen binding polypeptide ofthe invention may exhibit a sequence identity of 80% or greater, 85% orgreater, 90% or greater, 95% or greater, 97% or greater, or up to 99% oreven 100% with a human VL domain across the framework regions FR1, FR2,FR3 and FR4, and in addition hypervariable loop L1 and hypervariableloop L2 may form a combination of predicted or actual canonical foldstructures which is the same as a canonical fold combination known tooccur naturally in the same human VL domain.

It is, of course, envisaged that VH domains exhibiting high sequenceidentity/sequence homology with human VH, and also structural homologywith hypervariable loops of human VH will be combined with VL domainsexhibiting high sequence identity/sequence homology with human VL, andalso structural homology with hypervariable loops of human VL to provideantigen binding polypeptides containing (camelid-derived) VH/VL pairingswith maximal sequence and structural homology to human-encoded VH/VLpairings. A particular advantage of the camelid platform provided by theinvention is that both the VH domain and the VL domain exhibit highsequence and structural homology with the variable domains of humanantibodies.

Structure of the Antigen Binding Polypeptide

The antigen binding polypeptide of the invention can take variousdifferent embodiments, provided that both a VH domain and a VL domainare present. Thus, in non-limiting embodiments the antigen bindingpolypeptide may be an immunoglobulin, an antibody or antibody fragment.The term “antibody” herein is used in the broadest sense andencompasses, but is not limited to, monoclonal antibodies (includingfull length monoclonal antibodies), polyclonal antibodies, multispecificantibodies (e.g., bispecific antibodies), so long as they exhibit theappropriate specificity for a target antigen. The term “monoclonalantibody” as used herein refers to an antibody obtained from apopulation of substantially homogeneous antibodies, i.e., the individualantibodies comprising the population are identical except for possiblenaturally occurring mutations that may be present in minor amounts.Monoclonal antibodies are highly specific, being directed against asingle antigenic site. Furthermore, in contrast to conventional(polyclonal) antibody preparations which typically include differentantibodies directed against different determinants (epitopes) on theantigen, each monoclonal antibody is directed against a singledeterminant or epitope on the antigen.

“Antibody fragments” comprise a portion of a full length antibody,generally the antigen binding or variable domain thereof. Examples ofantibody fragments include Fab, Fab′, F(ab′)2, bi-specific Fab's, and Fvfragments, diabodies, linear antibodies, single-chain antibodymolecules, a single chain variable fragment (scFv) and multispecificantibodies formed from antibody fragments (see Holliger and Hudson,Nature Biotechnol. 23:1126-36 (2005), the contents of which areincorporated herein by reference).

In non-limiting embodiments, antibodies and antibody fragments accordingto the invention may comprise CH1 domains and/or CL domains, the aminoacid sequence of which is fully or substantially human. Where theantigen binding polypeptide of the invention is an antibody intended forhuman therapeutic use, it is typical for the entire constant region ofthe antibody, or at least a part thereof, to have fully or substantiallyhuman amino acid sequence. Therefore, an antibody of the invention mustcomprise VH and VL domains, at least one of which includes at least onehypervariable loop derived from Camelidae, but one or more or anycombination of the CH1 domain, hinge region, CH2 domain, CH3 domain andCL domain (and CH4 domain if present) may be fully or substantiallyhuman with respect to it's amino acid sequence.

Advantageously, the CH1 domain, hinge region, CH2 domain, CH3 domain andCL domain (and CH4 domain if present) may all have fully orsubstantially human amino acid sequence. In the context of the constantregion of a humanised or chimeric antibody, or an antibody fragment, theterm “substantially human” refers to an amino acid sequence identity ofat least 90%, or at least 95%, or at least 97%, or at least 99% with ahuman constant region. The term “human amino acid sequence” in thiscontext refers to an amino acid sequence which is encoded by a humanimmunoglobulin gene, which includes germline, rearranged and somaticallymutated genes. The invention also contemplates polypeptides comprisingconstant domains of “human” sequence which have been altered, by one ormore amino acid additions, deletions or substitutions with respect tothe human sequence.

As discussed elsewhere herein, it is contemplated that one or more aminoacid substitutions, insertions or deletions may be made within theconstant region of the heavy and/or the light chain, particularly withinthe Fc region. Amino acid substitutions may result in replacement of thesubstituted amino acid with a different naturally occurring amino acid,or with a non-natural or modified amino acid. Other structuralmodifications are also permitted, such as for example changes inglycosylation pattern (e.g. by addition or deletion of N- or O-linkedglycosylation sites). Depending on the intended use of the antibody, itmay be desirable to modify the antibody of the invention with respect toits binding properties to Fc receptors, for example to modulate effectorfunction. For example cysteine residue(s) may be introduced in the Fcregion, thereby allowing interchain disulfide bond formation in thisregion. The homodimeric antibody thus generated may have improvedinternalization capability and/or increased complement-mediated cellkilling and antibody-dependent cellular cytotoxicity (ADCC). See Caronet al., J. Exp. Med. 176:1191-1195 (1992) and Shopes, B. J. Immunol.148:2918-2922 (1992). Alternatively, an antibody can be engineered whichhas dual Fc regions and may thereby have enhanced complement lysis andADCC capabilities. See Stevenson et al., Anti-Cancer Drug Design3:219-230 (1989). The invention also contemplates immunoconjugatescomprising an antibody as described herein conjugated to a cytotoxicagent such as a chemotherapeutic agent, toxin (e.g., an enzymaticallyactive toxin of bacterial, fungal, plant or animal origin, or fragmentsthereof), or a radioactive isotope (i.e., a radioconjugate). Fc regionsmay also be engineered for half-life extension.

The invention can, in certain embodiments, encompass chimericCamelidae/human antibodies, and in particular chimeric antibodies inwhich the VH and VL domains are of fully camelid sequence (e.g. Llama oralpaca) and the remainder of the antibody is of fully human sequence. Inpreferred embodiments the invention also encompasses “humanised” or“germlined” Camelidae antibodies, and Camelidae/human chimericantibodies, in which the VH and VL domains contain one or more aminoacid substitutions in the framework regions in comparison to CamelidaeVH and VL domains obtained by active immunisation. Such “humanisation”increases the % sequence identity with human germline VH or VL domainsby replacing mis-matched amino acid residues in a starting Camelidae VHor VL domain with the equivalent residue found in a humangermline-encoded VH or VL domain.

The invention still further encompasses CDR-grafted antibodies in whichCDRs (or hypervariable loops) derived from a Camelidae antibody, forexample an Camelidae antibody raised by active immunisation with atarget antigen, or otherwise encoded by a camelid gene, are grafted ontoa human VH and VL framework, with the remainder of the antibody alsobeing of fully human origin. However, given the high degree of aminoacid sequence homology and structural homology they have observedbetween Camelidae and human immunoglobulins, the inventors anticipatethat in the majority of cases it will be possible to achieve the levelsof human homology required for in vivo therapeutic use via“humanisation” of the framework regions of camelid-derived VH and VLdomains without the need for CDR grafting or via CDR grafting on tolimited number of backbone sequences without the need for veneering(also see Almagro et al, Frontiers in Bioscience 13: 1619-1633 (2008),the contents of which are incorporated herein by reference).

Humanised, chimeric and CDR-grafted antibodies according to theinvention, particularly antibodies comprising hypervariable loopsderived from active immunisation of Camelidae with a target antigen, canbe readily produced using conventional recombinant DNA manipulation andexpression techniques, making use of prokaryotic and eukaryotic hostcells engineered to produce the polypeptide of interest and includingbut not limited to bacterial cells, yeast cells, mammalian cells, insectcells, plant cells, some of them as described herein and illustrated inthe accompanying examples.

The invention also encompasses antigen binding polypeptides whereineither one or other of the VH or VL domain is obtained from Camelidae,or contains at least one CDR or hypervariable region derived fromCamelidae, and the “other” variable domain has non-camelid, e.g. human,amino acid sequence. Thus, it is contemplated to pair a camelid VHdomain with a human VL domain, or to pair a human VH domain with acamelid VL domain. Such pairings may increase the availableantigen-binding repertoire from which to select high affinity binderswith the desired antigen binding properties.

The invention still further extends to antigen binding polypeptideswherein the hypervariable loop(s) or CDR(s) of the VH domain and/or theVL domain are obtained from Camelidae, but wherein at least one of said(camelid-derived) hypervariable loops or CDRS has been engineered toinclude one or more amino acid substitutions, additions or deletionsrelative to the camelid-encoded sequence. Such changes include“humanisation” of the hypervariable loops/CDRs. Camelid-derived HVs/CDRswhich have been engineered in this manner may still exhibit an aminoacid sequence which is “substantially identical” to the amino acidsequence of a camelid-encoded HV/CDR. In this context, “substantialidentity” may permit no more than one, or no more than two amino acidsequence mis-matches with the camelid-encoded HV/CDR.

Antibodies according to the invention may be of any isotype. Antibodiesintended for human therapeutic use will typically be of the IgA, IgD,IgE IgG, IgM type, often of the IgG type, in which case they can belongto any of the four sub-classes IgG1, IgG2a and b, IgG3 or IgG4. Withineach of these sub-classes it is permitted to make one or more amino acidsubstitutions, insertions or deletions within the Fc portion, or to makeother structural modifications, for example to enhance or reduceFc-dependent functionalities.

Antigen binding polypeptides according to the invention may be useful ina wide range of applications, both in research and in the diagnosisand/or treatment of diseases. Because of the high degree of amino acidsequence identity with the VH and VL domains of natural humanantibodies, and the high degree of structural homology (specifically thecorrect combinations of canonical folds as are found in humanantibodies) the antigen binding polypeptides of the invention,particularly in the form of monoclonal antibodies, will find particularutility as human therapeutic agents.

The invention provides a platform for production of antigen bindingpolypeptides, and specifically monoclonal antibodies, against a widerange of antigens and in its broadest aspect the invention is notintended to be limited with respect to the exact identity of the targetantigen, nor indeed the specificity or affinity of binding to the targetantigen. However, in particular, non-limiting, embodiments the targetantigen may be a non-camelid antigen, a bacterial antigen, a viralantigen or a human antigen. In a preferred embodiment the target antigenmay be an antigen of particular therapeutic importance. The term “targetof therapeutic importance” refers to a target involved in formation,onset, progression, mediation of human or animal diseases or of theeffects related to the respective disease. Included within thisdefinition are targets wherein the expression levels and/or activity ofthe target are modulated by antibody binding (e.g. receptors whoseactivity may be modulated by binding of agonist or antagonistantibodies), and targets wherein the activity and/or expression of thetarget has a direct or indirect impact on a disease.

By way of example, “human antigens” may include naturally occurringhuman polypeptides (proteins) which function as receptors, receptorligands, cell-signalling molecules, hormones, cytokines or cytokinereceptors, neurotransmitters, etc. By “naturally occurring” is meantthat the polypeptide is expressed within the human body, at any stage ifits development, including polypeptides expressed by the human bodyduring the course of a disease.

Non-limiting embodiments of the antigen binding polypeptide of theinvention include the following:

A chimeric antigen binding polypeptide comprising a VH domain and a VLdomain, wherein at least one hypervariable loop or complementaritydetermining region (CDR) in the VH domain or the VL domain is obtainedfrom a VH or VL domain of a species in the family Camelidae. In aparticular embodiment both the VH domain and the VL domain are obtainedfrom Llama (Lama glama).

A recombinantly expressed antigen binding polypeptide comprising a VHdomain and a VL domain, wherein at least one hypervariable loop orcomplementarity determining region (CDR) in the VH domain or the VLdomain is obtained from a VH or VL domain of a species in the familyCamelidae. In a particular embodiment both the VH domain and the VLdomain are obtained from Llama (Lama glama).

A monoclonal antibody comprising a VH domain and a VL domain, wherein atleast one hypervariable loop or complementarity determining region (CDR)in the VH domain or the VL domain is obtained from a VH or VL domain ofa species in the family Camelidae. In a particular embodiment both theVH domain and the VL domain are obtained from Llama (Lama glama).

An antigen binding polypeptide comprising a VH domain and a VL domain,wherein at least one hypervariable loop or complementarity determiningregion (CDR) in the VH domain or the VL domain is obtained from a VH orVL domain of a species in the family Camelidae and wherein said antigenbinding polypeptide is immunoreactive with a target antigen oftherapeutic or diagnostic importance. In a particular embodiment boththe VH domain and the VL domain are obtained from Llama (Lama glama).

A chimeric antigen binding polypeptide comprising or consisting of a VHdomain of a conventional antibody of a camelid (in particular Llama oralpaca), a VL domain of a conventional antibody of a camelid (inparticular Llama or alpaca) and one or more constant domains of a humanantibody. In a particular embodiment both the VH domain and the VLdomain are obtained from Llama (Lama glama).

A chimeric antigen binding polypeptide immunoreactive with a targetantigen of therapeutic or diagnostic importance, which antigen bindingpolypeptide comprises or consists of a VH domain of a conventionalantibody of a camelid (in particular Llama or alpaca), a VL domain of aconventional antibody of a camelid (in particular Llama or alpaca) andone or more constant domains of a human antibody. In a particularembodiment both the VH domain and the VL domain are obtained from Llama(Lama glama).

A chimeric antibody comprising or consisting of a VH domain of aconventional antibody of a camelid (in particular Llama or alpaca), a VLdomain of a conventional antibody of a camelid (in particular Llama oralpaca) and the constant domains of a human antibody of an isotypeselected from the group consisting of: IgG, IgM, IgD, IgE and IgA. In aparticular embodiment both the VH domain and the VL domain are obtainedfrom Llama (Lama glama).

A chimeric antigen binding polypeptide immunoreactive with a targetantigen of therapeutic or diagnostic importance, which antigen bindingpolypeptide comprises or consists of a VH domain of a conventionalantibody of a camelid (in particular Llama or alpaca), a VL domain of aconventional antibody of a camelid (in particular Llama or alpaca) andthe constant domains of a human antibody of an isotype selected from thegroup consisting of: IgG, IgM, IgD, IgE, IgA. In a particular embodimentboth the VH domain and the VL domain are obtained from Llama (Lamaglama).

In particular embodiments of the foregoing, both the VH and the VLdomain may be from the same species of camelid (in particular Llama oralpaca), and may even be from the same animal within this species, forexample a single animal which has been actively immunised. Inparticular, both the VH domain and the VL domain may be obtained from asingle actively immunised Llama. However, it is not excluded that the VHand VL domain may be obtained from different animals, or non-immunelibraries.

In the foregoing embodiments, the terms “VH domain of a conventionalantibody of a camelid” and “VL domain of a conventional antibody of acamelid” are intended to encompass variants which have been engineeredto introduce one or more changes in amino acid sequence, such asvariants which have been “humanised” or “germlined” in one or moreframework regions, as described elsewhere herein, and also encompass theproducts of synthetic (e.g. codon-optimised) genes, as describedelsewhere herein.

Polynucleotides, Vectors and Recombinant Expression

The invention also provides a polynucleotide molecule encoding theantigen binding polypeptide of the invention, an expression vectorcontaining a nucleotide sequence encoding the antigen bindingpolypeptide of the invention operably linked to regulatory sequenceswhich permit expression of the antigen binding polypeptide in a hostcell or cell-free expression system, and a host cell or cell-freeexpression system containing this expression vector.

Polynucleotide molecules encoding the antigen binding polypeptide of theinvention include, for example, recombinant DNA molecules.

The terms “nucleic acid”, “polynucleotide” or a “polynucleotidemolecule” as used herein interchangeably and refer to any DNA or RNAmolecule, either single- or double-stranded and, if single-stranded, themolecule of its complementary sequence. In discussing nucleic acidmolecules, a sequence or structure of a particular nucleic acid moleculemay be described herein according to the normal convention of providingthe sequence in the 5′ to 3′ direction. In some embodiments of theinvention, nucleic acids or polynucleotides are “isolated.” This term,when applied to a nucleic acid molecule, refers to a nucleic acidmolecule that is separated from sequences with which it is immediatelycontiguous in the naturally occurring genome of the organism in which itoriginated. For example, an “isolated nucleic acid” may comprise a DNAmolecule inserted into a vector, such as a plasmid or virus vector, orintegrated into the genomic DNA of a prokaryotic or eukaryotic cell ornon-human host organism. When applied to RNA, the term “isolatedpolynucleotide” refers primarily to an RNA molecule encoded by anisolated DNA molecule as defined above. Alternatively, the term mayrefer to an RNA molecule that has been purified/separated from othernucleic acids with which it would be associated in its natural state(i.e., in cells or tissues). An isolated polynucleotide (either DNA orRNA) may further represent a molecule produced directly by biological orsynthetic means and separated from other components present during itsproduction.

For recombinant production of an antigen binding polypeptide accordingto the invention, a recombinant polynucleotide encoding it may beprepared (using standard molecular biology techniques) and inserted intoa replicable vector for expression in a chosen host cell, or a cell-freeexpression system. Suitable host cells may be prokaryote, yeast, orhigher eukaryote cells, specifically mammalian cells. Examples of usefulmammalian host cell lines are monkey kidney CV1 line transformed by SV40(COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cellssubcloned for growth in suspension culture, Graham et al., J. Gen.Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10);Chinese hamster ovary cells/−DHFR(CHO, Urlaub et al., Proc. Natl. Acad.Sci. USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol.Reprod. 23:243-251 (1980)); mouse myeloma cells SP2/0-AG14 (ATCC CRL1581; ATCC CRL 8287) or NS0 (HPA culture collections no. 85110503);monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells(VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells(BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); humanliver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCCCCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68(1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2), aswell as DSM's PERC-6 cell line. Expression vectors suitable for use ineach of these host cells are also generally known in the art.

It should be noted that the term “host cell” generally refers to acultured cell line. Whole human beings into which an expression vectorencoding an antigen binding polypeptide according to the invention hasbeen introduced are explicitly excluded from the scope of the invention.

In an important aspect, the invention also provides a method ofproducing a recombinant antigen binding polypeptide which comprisesculturing a host cell (or cell free expression system) containingpolynucleotide (e.g. an expression vector) encoding the recombinantantigen binding polypeptide under conditions which permit expression ofthe antigen binding polypeptide, and recovering the expressed antigenbinding polypeptide. This recombinant expression process can be used forlarge scale production of antigen binding polypeptides according to theinvention, including monoclonal antibodies intended for humantherapeutic use. Suitable vectors, cell lines and production processesfor large scale manufacture of recombinant antibodies suitable for invivo therapeutic use are generally available in the art and will be wellknown to the skilled person.

Further aspects of the invention relate to test kits, includingdiagnostic kits etc. comprising an antigen binding polypeptide accordingto the invention, and also pharmaceutical formulations comprising anantigen binding polypeptide according to the invention.

Where the antigen binding polypeptide is intended for diagnostic use,for example where the antigen binding polypeptide is specific for anantigen which is a biomarker of a disease state or a diseasesusceptibility, then it may be convenient to supply the antigen bindingpolypeptide as a component of a test kit. Diagnostic tests typicallytake the form of standard immunoassays, such as ELISA, radioimmunoassay,Elispot, etc. The components of such a test kit may vary depending onthe nature of the test or assay it is intended to carry out using theantigen binding polypeptide of the invention, but will typically includeadditional reagents required to carry out an immunoassay using theantigen binding polypeptide of the invention. Antigen bindingpolypeptides for use as diagnostic reagents may carry a revealing label,such as for example a fluorescent moiety, enzymatic label, orradiolabel.

Antigen binding polypeptides intended for in vivo therapeutic use aretypically formulated into pharmaceutical dosage forms, together with oneor more pharmaceutically acceptable diluents, carriers or excipients(Remington's Pharmaceutical Sciences, 16th edition., Osol, A. Ed. 1980).Antigen binding polypeptides according to the invention are typicallyformulated as sterile aqueous solutions, to be administeredintravenously, or by intramuscular, intraperitoneal,intra-cerebrospinal, intratumoral, oral, peritumoral, subcutaneous,intra-synovial, intrathecal, topical, sublingual or inhalation routes,to a mammalian subject, typically a human patient, in need thereof. Forthe prevention or treatment of disease, the appropriate dosage ofantigen binding polypeptide will depend on the type of disease to betreated, the severity and clinical course of the disease, plus thepatient's age, weight and clinical history, and will be determined bythe judgement of the attending physician.

Processes for the Production of Antigen Binding Polypeptides

A key aspect of the present invention relates to processes for theproduction of high affinity antigen binding polypeptides, andspecifically monoclonal antibodies, against a target antigen ofinterest.

Accordingly, the invention provides a process for preparing an antigenbinding polypeptide immunoreactive with a target antigen, said processcomprising:

-   -   (a) determining the nucleotide sequence encoding at least one        hypervariable loop or complementarity determining region (CDR)        of the VH and/or the VL domain of a Camelidae conventional        antibody immunoreactive with said target antigen; and    -   (b) expressing an antigen binding polypeptide immunoreactive        with said target antigen, said antigen binding polypeptide        comprising a VH and a VL domain, wherein at least one        hypervariable loop or complementarity determining region (CDR)        of the VH domain or the VL domain has an amino acid sequence        encoded by the nucleotide sequence determined in part (a).

In one embodiment, the antigen binding polypeptide expressed in part (b)is not identical to the Camelidae conventional antibody of part (a).

In one non-limiting embodiment, the invention provides a process forpreparing a recombinant antigen binding polypeptide that isimmunoreactive with (or specifically binds to) a target antigen, said anantigen binding polypeptide comprising a VH domain and a VL domain,wherein at least one hypervariable loop or complementarity determiningregion (CDR) in the VH domain or the VL domain is obtained from aspecies in the family Camelidae, said process comprising the steps of:

-   -   (a) isolating Camelidae nucleic acid encoding at least one        hypervariable loop or complementarity determining region (CDR)        of the VH and/or the VL domain of a Camelidae conventional        antibody immunoreactive with said target antigen;    -   (b) preparing a recombinant polynucleotide comprising a        nucleotide sequence encoding hypervariable loop(s) or        complementarity determining region(s) having amino acid sequence        identical to the hypervariable loop(s) or complementarity        determining region(s) encoded by the nucleic acid isolated in        step (a), which recombinant polynucleotide encodes an antigen        binding polypeptide comprising a VH domain and a VL domain that        is immunoreactive with (or specifically binds to) said target        antigen; and    -   (c) expressing said antigen binding polypeptide from the        recombinant polynucleotide of step (b).

In one embodiment, the antigen binding polypeptide expressed in part (c)is not identical to the Camelidae conventional antibody of part (a).

The foregoing methods may be referred to herein as “general processes”for preparing antigen binding polypeptides.

The first step of either process may involve active immunisation of aspecies in the family Camelidae in order to elicit an immune responseagainst the target antigen, thereby raising camelid conventionalantibodies immunoreactive with the target antigen. Protocols forimmunisation of camelids are described in the accompanying examples. Theantigen preparation used for immunisation may be a purified form of thetarget antigen, for example recombinantly expressed polypeptide, or animmunogenic fragment thereof. However, it is also possible to immunisewith crude preparations of the antigen, such as like isolated cells ortissue preparations expressing or encoding the target antigen, celllysates, cell supernatants or fractions such as cell membranes, etc., orwith, a polynucleotide encoding said target antigen (a DNAimmunisation).

The process will typically involve immunisation of animals of aCamelidae species (including, but limited to, llamas and alpacas), andadvantageously these animals will belong to an outbred population.However, it is also contemplated to use transgenic animals (e.g.transgenic mice) containing the Camelid conventional Ig locus, or atleast a portion thereof.

A topic of increasing interest seems to be the difference between thecomplementarity determining regions (CDRs) of in vivo and in vitrogenerated antibodies. The inventors surmise that the in vivo selectionhas a favourable impact on the immunogenicity, functionality, stabilityand therefore improved manufacturability of the resulting antibodies,whilst synthetic CDRs generated and selected in vitro may have adisadvantage from this point of view. This is important since a giventherapeutic antibody risks to be neutralized by the so calledanti-idiotypic antibody response from the patient (Lonberg, NatureBiotechnology, 23: 1117-1125, (2005)).

A key advantage of processes according to the invention based on activeimmunisation of camelids stems from the fact that all species ofCamelidae can be maintained in large outbred populations where theindividual animals have a different genetic background. It is thereforepossible to use active immunisation to elicit a strong and diverseimmune response against the antigen of interest from which a diversepool of potential antigen binding molecules can be obtained. Asillustrated in the accompanying examples, the present inventors haveobserved that active immunisation of camelids can generate Fab fragmentsbinding to a target antigen with a high degree of immunodiversity.Without wishing to be bound by theory, the inventors surmise that thephylogenetic distance between humans and camelids may be important forproduction of a diverse immune response against a given target antigen.In contrast, the non-human primates are phylogenetically close tohumans, thus targets with high homology between non-human primates andhumans may elicit only a limited immune response in terms of strengthand diversity in non-human primates.

The ability to use active immunisation in an outbred population which isphylogenetically distant from human would not be particularlyadvantageous if the antibodies so-produced were to exhibit a lowsequence and structural homology with human antibodies such thatsubstantial “protein engineering” would be required to create acandidate antibody with therapeutic potential. It is therefore extremelyimportant that the inventors have shown that the Camelidae germline (andsomatically mutated sequences) encodes both VH and VL domains with avery high degree of sequence and structural homology with human VH andVL domains (as explained above). This high degree of homology incombination with the availability of large outbred populations resultsin a very powerful platform for development of monoclonal antibodies foruse as human therapeutics.

Following active immunisation with the target antigen, peripheral bloodlymphocytes or biopsies such as lymph nodes or spleen biopsies may beisolated from the immunised animal and screened for production ofconventional camelid antibodies against the target antigen. Techniquessuch as enrichment using panning or FACS sorting may be used at thisstage to reduce the complexity of the B cell repertoire to be screened,as illustrated in the examples. Antigen-specific B cells are thenselected and used for total RNA extraction and subsequent cDNAsynthesis. Nucleic acid encoding the native camelid VH and VL domains(specific for the target antigen) can be isolated by PCR.

It is not essential to use active immunisation in order to identifycamelid convention antibodies immunoreactive with a target of interest.In other embodiments it may be possible to make use of the camelid's ownimmune response, either the immunodiversity naturally present in theanimal, or for example a diseased animal or animal which has beennaturally exposed to a particular pathogen, e.g. by normal infectionroutes. In this regard, the invention encompasses the use of non-immunelibraries. If “natural” immune responses within the camelid already giverise to antibodies which bind the target antigen of interest, then it ispossible to make use of the genetic engineering techniques describedherein, and other standard techniques known in the art, in order toculture and isolate B cells producing such antibodies, or producemonoclonal cultures of such antibodies, and/or to determine thenucleotide sequence of the camelid gene segments encoding the VH and/orVL domains of such antibodies. Armed with this sequence information, itis then possible to engineer recombinant DNA constructs encoding antigenbinding polypeptides which embody the camelid derived VH and/or VL, orthe hypervariable loops (or CDRs) thereof.

Nucleic acid encoding camelid VH and VL domains (whether obtained byactive immunisation or by other means) may be cloned directly into anexpression vector for the production of an antigen binding polypeptideaccording to the invention. In particular, these sequences could becloned into an expression vector which also encodes a human antibodyconstant region, or a portion thereof, in order to produce a chimericantibody. However, it is typical to carry out further manipulations onthe isolated camelid VH and VL sequences before cloning and expressionwith human constant region sequences.

As a first step, candidate camelid VH and VL sequences (includingsequences isolated following the active immunisation) may be used toprepare a camelid libraries (e.g. Fab libraries, as described in theaccompanying examples). The library may then be screened (e.g. usingphage display) for binding to the target antigen. Promising leadcandidates can be further tested for target antigen binding, for exampleusing Biacore or a suitable bioassay. Finally, the sequences encodingthe VH and VL domains of the most promising leads can be cloned as anin-frame fusion with sequences encoding a human antibody constantregion.

It is not essential that the polynucleotide sequence used to encode the(camelid-derived) HVs/CDRs (e.g. for recombinant expression of theantigen binding polypeptide of the invention) is identical to the nativepolynucleotide sequence which naturally encodes the HVs/CDRs in thecamelid. Therefore, the invention encompasses/permits codonoptimisation, and other changes in polynucleotide sequence related tocloning and/or expression, which do not alter the encoded amino acidsequence.

In certain embodiments, “chain shuffling” may be performed in which aparticular variable domain known to bind the antigen of interest ispaired with each of a set of variable domains of the opposite type (i.e.VH paired with VL library or vice versa), to create libraries, and theresulting “promiscuous” combinations of VH/VL tested for antigen bindingaffinity and/or specificity. Alternatively, a library of VH domainscould be paired with a library of VL domains, either randomly or in ahierarchical manner, and the resulting combinations tested (see Clacksonet al., Nature., Vol. 352. pp 624-638, 1991). In this process, thelibraries may be libraries of rearranged VH and VL (Vκ or Vλ) fromcamelids which display immunity to the antigen of interest (includinganimals which have been actively immunised). The chain shuffling processcan increase immunodiversity and produce pairings with significantlyenhanced affinity.

The invention also contemplates performing epitope imprinted selection(so-called “guided selection”) starting from a camelid VH or VL domain,wherein the other variable domain is taken from a non-camelid species,e.g. human. Thus, in one embodiment a camelid VH domain may be“shuffled” with a library of human-encoded VL domains, to replace thenative camelid-encoded VL domain, resulting in camelid VH/human VLpairings. One or more of these pairings may then be subjected a secondchain shuffling step in which the human VL domain is shuffled against alibrary of VH domains, which may be human-encoded. This second step mayproduce human-encoded VH/VL combinations which have the epitope imprintof the original camelid-encoded VH/VL combination.

Also included within the scope of the invention is the reverse “chainshuffling” process, starting with non-camelid (preferably human)-encodedVH/VL domain combination which binds to an antigen of interest. Thiscould be, for example, a fully human therapeutic antibody against avalidated disease target. Starting from this VH/VL combination, it ispossible to carry out a first round of selection in which the VH domainis “shuffled” with a library of camelid-encoded VL domains (or viceversa), and the pairings tested for antigen binding. Selectednon-camelid (e.g. human) VH/camelid VL pairings may then be subjected toa second round of selection in which the camelid-encoded VL is shuffledagainst a library of camelid-encoded VH, and the resulting pairingstested for antigen binding. As a result, it may be possible to produce acamelid VH/camelid VL combination which carries the epitope imprint ofthe starting VH/VL combination. This camelid VH/VL combination could befurther engineered/modified and combined with human-encoded constantdomains as required, using any of the processes described herein.

In the processes of the invention, “native” camelid-derived VH and VLdomains may be subject to protein engineering in which one or moreselective amino acid substitutions are introduced, typically in theframework regions. The reasons for introducing such substitutions intothe “wild type” camelid sequence can be (i) humanisation of theframework region, (ii) improvement in stability, bioavailability,product uniformity, tissue penetration, etc., or (iii) optimisation oftarget antigen binding.

“Humanisation” of camelid-derived VH and VL domains by selectivereplacement of one or more amino acid residues in the framework regionsmay be carried out according to well-established principles (asillustrated in the accompanying examples, and reviewed by Almagro et al.Frontiers in Bioscience 13:1619-1633 (2008), the contents of which arespecifically incorporated herein by reference). It will be appreciatedthat the precise identity of the amino acid changes made to achieveacceptable “humanisation” of any given VH domain, VL domain orcombination thereof will vary on a case-by-case basis, since this willdepend upon the sequence of the framework regions derived from Camelidaeand the starting homology between these framework regions and theclosest aligning human germline (or somatically mutated) frameworkregion, and possible also on the sequence and conformation of thehypervariable loops which form the antigen binding site.

The overall aim of humanisation is to produce a molecule in which the VHand VL domains exhibit minimal immunogenicity when introduced into ahuman subject, whilst retaining the specificity and affinity of theantigen binding site formed by the parental VH and VL domains encoded byCamelidae (e.g. camelid VH/VL obtained by active immunisation). Thereare a number of established approaches to humanisation which, can beused to achieve this aim. Techniques can be generally classified aseither rational approaches or empirical approaches. Rational approachesinclude CDR-grafting, resurfacing or veneering, superhumanization andhuman string content optimisation. Empirical approaches include the FRlibrary approach, guided selection, FR shuffling and humaneering. All ofthese techniques are reviewed in Almagro, Frontiers in Bioscience 2008,ibid. and any of these techniques, or combinations or modificationsthereof, can be used to prepare “humanised” antigen binding polypeptidesaccording to the invention.

Methods of Library Construction

In a related aspect, the invention also encompasses a method ofproducing a library of expression vectors encoding VH and/or VL domainsof camelid conventional antibodies, said method comprising the steps:

a) amplifying regions of nucleic acid molecules encoding VH and/or VLdomains of camelid conventional antibodies to obtain amplified genesegments, each gene segment containing a sequence of nucleotidesencoding a VH domain or a sequence of nucleotides encoding a VL domainof a camelid conventional antibody, andb) cloning the gene segments obtained in a) into expression vectors,such that each expression vector contains at least a gene segmentencoding a VH domain and/or a gene segment encoding a VL domain, wherebya library of expression vectors is obtained.

The above methods of “library construction” may also form part of thegeneral process for production of antigen binding polypeptides of theinvention, described above. Hence, any feature described as beingpreferred or advantageous in relation to this aspect of the inventionmay also be taken as preferred or advantageous in relation to thegeneral process, and vice versa, unless otherwise stated.

In one embodiment, the nucleic acid amplified in step a) comprises cDNAor genomic DNA prepared from lymphoid tissue of a camelid, said lymphoidtissue comprising one or more B cells, lymph nodes, spleen cells, bonemarrow cells, or a combination thereof. Circulating B cells areparticularly preferred. The present inventors have surprisingly foundthat peripheral blood lymphocytes (PBLs) can be used as a source ofnucleic acid encoding VH and VL domains of conventional camelidantibodies, i.e. there is sufficient quantity of plasma cells(expressing antibodies) present in a sample of PBLs to enable directamplification. This is advantageous because PBLs can be prepared from awhole blood sample taken from the animal (camelid). This avoids the needto use invasive procedures to obtain tissue biopsies (e.g. from spleenor lymph node), and means that the sampling procedure can be repeated asoften as necessary, with minimal impact on the animal. For example, itis possible to actively immunise the camelid, remove a first bloodsample from the animal and prepare PBLs, then immunise the same animal asecond time, either with a “boosting” dose of the same antigen or with adifferent antigen, then remove a second blood sample and prepare PBLs.

Accordingly, a particular embodiment of this method of the invention mayinvolve: preparing a sample containing PBLs from a camelid, preparingcDNA or genomic DNA from the PBLs and using this cDNA or genomic DNA asa template for amplification of gene segments encoding VH or VL domainsof camelid conventional antibodies.

In one embodiment the lymphoid tissue (e.g. circulating B cells) isobtained from a camelid which has been actively immunised, as describedelsewhere herein. However, this embodiment is non-limiting and it isalso contemplated to prepare non-immune libraries and libraries derivedfrom lymphoid tissue of diseased camelids, also described elsewhereherein.

Conveniently, total RNA (or mRNA) can be prepared from the lymphoidtissue sample (e.g. peripheral blood cells or tissue biopsy) andconverted to cDNA by standard techniques. It is also possible to usegenomic DNA as a starting material.

This aspect of the invention encompasses both a diverse libraryapproach, and a B cell selection approach for construction of thelibrary. In a diverse library approach, repertoires of VH andVL-encoding gene segments may be amplified from nucleic acid preparedfrom lymphoid tissue without any prior selection of B cells. In a B cellselection approach, B cells displaying antibodies with desiredantigen-binding characteristics may be selected, prior to nucleic acidextraction and amplification of VH and VL-encoding gene segments.

Various conventional methods may be used to select camelid B cellsexpressing antibodies with desired antigen-binding characteristics. Forexample, B cells can be stained for cell surface display of conventionalIgG with fluorescently labelled monoclonal antibody (mAb, specificallyrecognizing conventional antibodies from llama or other camelids) andwith target antigen labelled with another fluorescent dye. Individualdouble positive B cells may then be isolated by FACS, and total RNA (orgenomic DNA) extracted from individual cells. Alternatively cells can besubjected to in vitro proliferation and culture supernatants withsecreted IgG can be screened, and total RNA (or genomic DNA) extractedfrom positive cells. In a still further approach, individual B cells maybe transformed with specific genes or fused with tumor cell lines togenerate cell lines, which can be grown “at will”, and total RNA (orgenomic DNA) subsequently prepared from these cells.

Instead of sorting by FACS, target specific B cells expressingconventional IgG can be “panned” on immobilized monoclonal antibodies(directed against camelid conventional antibodies) and subsequently onimmobilized target antigen. RNA (or genomic DNA) can be extracted frompools of antigen specific B cells or these pools can be transformed andindividual cells cloned out by limited dilution or FACS.

B cell selection methods may involve positive selection, or negativeselection.

Whether using a diverse library approach without any B cell selection,or a B cell selection approach, nucleic acid (cDNA or genomic DNA)prepared from the lymphoid tissue is subject to an amplification step inorder to amplify gene segments encoding individual VH domains or VLdomains.

Total RNA extracted from the lymphoid tissue (e.g. peripheral B cells ortissue biopsy) may be converted into random primed cDNA or oligo dTprimer can be used for cDNA synthesis, alternatively Ig specificoligonucleotide primers can be applied for cDNA synthesis, or mRNA (i.e.poly A RNA) can be purified from total RNA with oligo dT cellulose priorto cDNA synthesis. Genomic DNA isolated from B cells can be used forPCR.

PCR amplification of heavy chain and light chain (kappa and lambda) genesegments encoding at least VH or VL can be performed with FR1 primersannealing to the 5′ end of the variable region in combination withprimers annealing to the 3′ end of CH1 or Ckappa/Clambda region with theadvantage that for these constant region primers only one primer isneeded for each type. This approach enables camelid Fabs to be cloned.Alternatively sets of FR4 primers annealing to the 3′ end of thevariable regions can be used, again for cloning as Fabs (fused to vectorencoded constant regions) or as scFv (single chain Fv, in which theheavy and light chain variable regions are linked via a flexible linkersequence); alternatively the variable regions can be cloned inexpression vectors allowing the production of full length IgG moleculesdisplayed on mammalian cells.

In general the amplification is performed in two steps; in the firststep with non-tagged primers using a large amount of cDNA (to maintaindiversity) and in the second step the amplicons are re-amplified in onlya few cycles with tagged primers, which are extended primers withrestriction sites introduced at the 5′ for cloning. Amplicons producedin the first amplification step (non-tagged primers) may be gel-purifiedto remove excess primers, prior to the second amplification step.Alternatively, promoter sequences may be introduced, which allowtranscription into RNA for ribosome display. Instead of restrictionsites recombination sites can be introduced, like the Cre-Lox or TOPOsites, that permit the site directed insertion into appropriate vectors.

Amplified gene segments encoding camelid conventional VH and VL domainsmay then be cloned into vectors suitable for expression of VH/VLcombinations as functional antigen binding polypeptides. By way ofexample, amplified VHCH1/VKCK/VLCL gene segments from pools of B cells(or other lymphoid tissue not subject to any B cell selection) may befirst cloned separately as individual libraries (primary libraries),then in a second step Fab or scFV libraries may be assembled by cuttingout the light chain fragments and ligating these into vectors encodingthe heavy chain fragments. The two step procedure supports thegeneration of large libraries, because the cloning of PCR products isrelatively inefficient (due to suboptimal digestion with restrictionenzymes). scFv encoding DNA fragments can be generated bysplicing-by-overlap extension PCR (SOE) based on a small overlap insequence in amplicons; by mixing VH and VL encoding amplicons with asmall DNA fragment encoding the linker in a PCR a single DNA fragment isformed due to the overlapping sequences.

Amplicons comprising VH and VL-encoding gene segments can be cloned inphage or phagemid vectors, allowing selection of target specificantibody fragments by using phage display based selection methods.Alternatively amplicons can be cloned into expression vectors whichpermit display on yeast cells (as Fab, scFv or full length IgG) ormammalian cells (as IgG).

In other embodiments, cloning can be avoided by using the amplicons forribosome display, in which a T7 (or other) promoter sequence andribosome binding site is included in the primers for amplification.After selection for binding to target antigen, pools are cloned andindividual clones are analyzed. In theory, larger immune repertoires canbe sampled using this approach as opposed to a phage display libraryapproach, because cloning of libraries and selection with phage islimited to 10¹⁰ to 10¹² clones.

When applying B cell sorting, amplicons contain VH or VL-encoding genesegments of individual target specific B cells can be cloned directlyinto bacterial or mammalian expression vectors for the production ofantibody fragments (scFVs or Fabs) or even full length IgG.

In a particular, non-limiting, embodiment of the “library construction”process, the invention provides a method of producing a library ofexpression vectors encoding VH and VL domains of camelid conventionalantibodies, said method comprising the steps:

a) actively immunising a camelid, thereby raising conventional camelidantibodies against a target antigen;

b) preparing cDNA or genomic DNA from a sample comprising lymphoidtissue (e.g. circulating B cells) from said immunised camelid(including, but not limited to, Llama or alpaca);

c) amplifying regions of said cDNA or genomic DNA to obtain amplifiedgene segments, each gene segment comprising a sequence of nucleotidesencoding a VH domain or a sequence of nucleotides encoding a VL domainof a camelid conventional antibody; and

d) cloning the gene segments obtained in c) into expression vectors,such that each expression vector contains a gene segment encoding a VHdomain and a gene segment encoding a VL domain and directs expression ofan antigen binding polypeptide comprising said VH domain and said VLdomain, whereby a library of expression vectors is obtained.

The foregoing methods may be used to prepare libraries ofcamelid-encoded VH and VL domains (in particular Llama and alpaca VH andVL domains), suitable for expression of VH/VL combinations as functionalantigen-binding polypeptides, e.g. in the form of scFVs, Fabs orfull-length antibodies.

Libraries of expression vectors prepared according to the foregoingprocess, and encoding camelid (including but not limited to Llama oralpaca) VH and VL domains, also form part of the subject-matter of thepresent invention.

In a particular embodiment the invention provides a library of phagevectors encoding Fab or scFV molecules, wherein each Fab or scFV encodedin the library comprises a VH domain of a camelid conventional antibodyand a VL domain of a camelid conventional antibody.

In one embodiment the library is a “diverse” library, in which themajority of clones in the library encode VH domains of unique amino acidsequence, and/or VL domains of unique amino acid sequence, includingdiverse libraries of camelid VH domains and camelid VL domains.Therefore, the majority (e.g. >90%) of clones in a diverse libraryencode a VH/VL pairing which differs from any other VH/VL pairingencoded in the same library with respect to amino acid sequence of theVH domain and/or the VL domain.

The invention also encompasses expression vectors containing VH andVL-encoding gene segments isolated from a single selected B cell of acamelid (e.g. Llama or alpaca).

In a further aspect, the present invention also provides a method ofselecting an expression vector encoding an antigen binding polypeptideimmunoreactive with a target antigen, the method comprising steps of:

i) providing a library of expression vectors, wherein each vector insaid library comprises a gene segment encoding a VH domain and a genesegment encoding a VL domain, wherein at least one of said VH domain orsaid VL domain is from a camelid conventional antibody, and wherein eachvector in said library directs expression of an antigen bindingpolypeptide comprising said VH domain and VL domain;ii) screening antigen binding polypeptides encoded by said library forimmunoreactivity with said target antigen, and thereby selecting anexpression vector encoding an antigen binding polypeptide immunoreactivewith said target antigen.

This method of the invention encompasses screening/selection of clonesimmunoreactive with target antigen, from a library of clones encodingVH/VL pairings. The method may also encompass library construction,which may be carried out using the library construction method describedabove. Optional downstream processing/optimisation steps may be carriedout on selected clones, as described below. This method of selection andscreening, may also form part of the general process for production ofantigen binding polypeptides of the invention, described above. Hence,any feature described as being preferred or advantageous in relation tothis aspect of the invention may also be taken as preferred oradvantageous in relation to the general process, and vice versa, unlessotherwise stated.

Screening and Selection of Clones Immunoreactive with Target Antigen

Screening/selection typically involves contacting expression productsencoded by clones in the library (ie. VH/VL pairings in the form ofantigen binding polypeptides, e.g. Fabs, scFVs or antibodies) with atarget antigen, and selecting one or more clones which encode a VH/VLpairings exhibiting the desired antigen binding characteristics.

Phage display libraries may be selected on immobilized target antigen oron soluble (often biotinylated) target antigen. The Fab format allowsaffinity driven selection due to its monomeric appearance and itsmonovalent display on phage, which is not possible for scFv (as aconsequence of aggregation and multivalent display on phage) and IgG(bivalent format). Two to three rounds of selections are typicallyneeded to get sufficient enrichment of target specific binders.

Affinity driven selections can be performed by lowering the amount oftarget antigen in subsequent rounds of selection, whereas extendedwashes with non-biotinylated target enables the identification ofbinders with extremely good affinities.

The selection procedure allows the user to home in on certain epitopes;whereas the classical method for elution of phage clones from theimmobilized target is based on a pH shock, which denatures the antibodyfragment and/or target, competition with a reference mAb against thetarget antigen or soluble receptor or cytokine leads to the elution ofphage displaying antibody fragments binding to the relevant epitope ofthe target (this is of course applicable to other display systems aswell, including the B cells selection method).

Individual clones taken from the selection outputs may be used for smallscale production of antigen-binding polypeptides (e.g. antibodyfragments) using periplasmic fractions prepared from the cells or theculture supernatants, into which the fragments “leaked” from the cells.Expression may be driven by an inducible promoter (e.g. the lacpromoter), meaning that upon addition of the inducer (IPTG) productionof the fragment is initiated. A leader sequence ensures the transport ofthe fragment into the periplasm, where it is properly folded and theintramolecular disulphide bridges are formed.

The resulting crude protein fractions may be used in target bindingassays, such as ELISA. For binding studies, phage prepared fromindividual clones can be used to circumvent the low expression yields ofFabs, which in general give very low binding signals. These proteinfractions can also be screened using in vitro receptor—ligand bindingassays to identify antagonistic antibodies; ELISA based receptor—ligandbinding assays can be used, also high throughput assays like Alphascreenare possible. Screening may be performed in radiolabelled ligand bindingassays, in which membrane fractions of receptor overexpressing celllines are immobilized; the latter assay is extremely sensitive, sinceonly picomolar amounts of radioactive cytokine are needed, meaning thatminute amounts of antagonistic Fabs present in the crude proteinfraction will give a positive read-out. Alternatively, FACS can beapplied to screen for antibodies, which inhibit binding of afluorescently labelled cytokine to its receptor as expressed on cells,while FMAT is the high throughput variant of this.

Fabs present in periplasmic fractions or partially purified by IMAC onits hexahistidine tag or by protein G (known to bind to the CH1 domainof Fabs) can be directly used in bioassays using cells, which are notsensitive to bacterial impurities; alternatively, Fabs from individualE. coli cells can be recloned in mammalian systems for the expression ofFabs or IgG and subsequently screened in bioassays.

Following identification of positive expression vector clones, i.e.clones encoding a functional VH/VL combination which binds to thedesired target antigen, it is a matter of routine to determine thenucleotide sequences of the variable regions, and hence deduce the aminoacid sequences of the encoded VH and VL domains.

If desired, the Fab (or scFV) encoding region may be recloned into analternative expression platform, e.g. a bacterial expression vector(identical to the phagemid vector, but without the gene 3 necessary fordisplay on phage), which allows larger amounts of the encoded fragmentto be produced and purified.

The affinity of target binding may be determined for the purified Fab(or scFV) by surface plasmon resonance (e.g. Biacore) or via othermethods, and the neutralizing potency tested using in vitroreceptor—ligand binding assays and cell based assays.

Families of antigen-binding, and especially antagonistic Fabs (or scFVs)may be identified on the basis of sequence analysis (mainly of VH, inparticular the length and amino acid sequence of CDR3 of the VH domain).

Potency Optimisation

Clones identified by screening/selection as encoding a VH/VL combinationwith affinity for the desired target antigen may, if desired, be subjectto downstream steps in which the affinity and/or neutralising potency isoptimised.

Potency optimization of the best performing member of each VH family canbe achieved via light chain shuffling, heavy chain shuffling or acombination thereof, thereby selecting the affinity variants naturallyoccurring in the animal. This is particularly advantageous inembodiments where the original camelid VH/VL domains were selected froman actively immunised camelid, since it is possible to perform chainshuffling using the original library prepared from the same immunisedanimal, thereby screening affinity variants arising in the sameimmunised animal.

For light chain shuffling the gene segment encoding the VH region (orVHCH1) of VH/VL pairing with desirable antigen binding characteristics(e.g. an antagonistic Fab) may be used to construct a library in whichthis single VH-encoding gene segment is combined with the light chainrepertoire of the library from which the clone was originally selected.For example, if the VH-encoding segment was selected from a library(e.g. Fab library) prepared from a camelid animal actively immunised toelicit an immune response against a target antigen, then the “chainshuffling” library may be constructed by combining this VH-encodingsegment with the light chain (VL) repertoire of the same immunisedcamelid. The resulting library may then be subject to selection of thetarget antigen, but under stringent conditions (low concentrations oftarget, extensive washing with non-biotinylated target in solution) toensure the isolation of the best affinity variant. Off-rate screening ofperiplasmic fractions may also assist in the identification of improvedclones. After sequence analysis and recloning into a bacterialproduction vector, purified selected Fabs may be tested for affinity(e.g. by surface plasmon resonance) and potency (e.g. by bioassay).

Heavy chain shuffling can be performed by cloning back the gene segmentencoding the light chain (VL) of a clone selected after light-chainshuffling into the original heavy chain library from the same animal(from which the original VH/VL-encoding clone was selected).Alternatively a CDR3 specific oligonucleotide primer can be used for theamplification of the family of VH regions, which can be cloned as arepertoire in combination with the light chain of the antagonistic Fab.Affinity driven selections and off-rate screening then allow theidentification of the best performing VH within the family.

It will be appreciated that the light chain shuffling and heavy chainshuffling steps may, in practice, be performed in either order, i.e.light chain shuffling may be performed first and followed by heavy chainshuffling, or heavy chain shuffling may be performed first and followedby light chain shuffling. Both possibilities are encompassed within thescope of the invention.

From light chains or heavy chains of VH/VL pairings (e.g. Fabs) withimproved affinity and potency the sequences of, in particular, the CDRscan be used to generate engineered variants in which mutations of theindividual Fabs are combined. It is known that often mutations can beadditive, meaning that combining these mutations may lead to an evenmore increased affinity.

Germlining and Formatting for Human Therapeutic Use

The VH and VL-encoding gene segments of selected expression clonesencoding VH/VL pairings exhibiting desirable antigen-bindingcharacteristics (e.g. phage clones encoding scFVs or Fabs) may besubjected to downstream processing steps and recloned into alternativeexpression platforms, such as vectors encoding antigen bindingpolypeptide formats suitable for human therapeutic use (e.g. full lengthantibodies with fully human constant domains).

Promising “lead” selected clones may be engineered to introduce one ormore changes in the nucleotide sequence encoding the VH domain and/orthe VL domain, which changes may or may not alter the encoded amino acidsequence of the VH domain and/or the VL domain. Such changes in sequenceof the VH or VL domain may be engineered for any of the purposesdescribed elsewhere herein, including germlining or humanisation, codonoptimisation, enhanced stability, optimal affinity etc.

The general principles germlining or humanisation described herein applyequally in this embodiment of the invention. By way of example, leadselected clones containing camelid-encoded VH and VL domains may begermlined/humanised in their framework regions (FRs) by applying alibrary approach. After alignment against the closest human germline(for VH and VL) and other human germlines with the identical canonicalfolds of CDR1 and CDR2, the residues to be changed in the FRs areidentified and the preferred human residue selected, as describedelsewhere herein in detail. Whilst germlining may involve replacement ofcamelid-encoded residues with an equivalent residue from the closestmatching human germline this is not essential, and residues from otherhuman germlines could also be used.

The germlining of a VH domain having an amino acid sequence homologousto a member of the human VH3 family will often involvereplacement/substitution of a number of residues, which already deviatein publically known Lama glama, Lama pacos or Camelus dromedariusderived germline sequences. Permitted amino acid substitutions forgermlining/humanisation of a VH3 domain of Lama glama, Lama pacos orCamelus dromedarius, and in particular Lama glama include, but are notlimited to, amino acid replacements at any one or any combination ofpositions 71, 83 and 84 in the framework region (using Kabat numbering).Such replacement(s) will involve substitution of the camelid-encodedresidue(s) at these positions with a different amino acid, which may bea natural or non-natural amino acid, and is preferably an amino acidknown to occur at the equivalent position in a human-encoded VH3 domain.For example, Alanine at position 71 might be replaced with serine oralanine, Lysine at position 83 might be replaced with Arginine andProline at position 84 might be replaced with Alanine. Accordingly,particular non-limiting embodiments of the antigen binding polypeptideof the invention include variants comprising a camelid (and morespecifically llama, alpaca or dromedary) VH domain exhibiting sequencehomology to a human VH3 domain, which VH domain includes amino acidsubstitutions (versus the camelid-encoded sequence) at one or more orall of positions 71, 83 and 84 (using Kabat numbering). In particular,variants with one or more or any combination of the followingsubstitutions are permitted: A changed to S at position 71, K changed toR at position 83 or P changed to A at position 84.

Once the amino acid sequences of the lead VH and VL domains (followingpotency optimisation, as appropriate) are known, synthetic genes of VHand VL can be designed, in which residues deviating from the humangermline are replaced with the preferred human residue (from the closestmatching human germline, or with residues occurring in other humangermlines, or even the camelid wild type residue). At this stage thegene segments encoding the variable domains may be re-cloned intoexpression vectors in which they are fused to human constant regions ofthe Fab, either during gene synthesis or by cloning in an appropriatedisplay vector.

The resulting VH and VL synthetic genes can be recombined into a Fablibrary or the germlined VH can be recombined with the wild type VL (andvice versa, referred to as “hybrid” libraries). Affinity-drivenselections will allow the isolation of the best performing germlinedversion, in case of the “hybrid” libraries, the best performinggermlined VH can be recombined with the best performing germlined VL.

Amino acid and nucleotide sequence information for the germlined Fabscan be used to generate codon-optimized synthetic genes for theproduction of full length human IgG of the preferred isotype (IgG1 forADCC and CDC, IgG2 for limited effector functions, IgG4 as for IgG2, butwhen monovalent binding is required). For non-chronic applications andacute indications bacterially or mammalian cell produced human Fab canproduced as well.

Combining steps of the above-described processes, in a particularnon-limiting embodiment the present invention provides a method ofproducing an expression vector encoding a chimeric antigen bindingpolypeptide immunoreactive with a target antigen, said method comprisingthe steps of:

a) actively immunising a camelid (including but not limited to Llama oralpaca), thereby raising conventional camelid antibodies against atarget antigen;

b) preparing cDNA or genomic DNA from a sample comprising lymphoidtissue (e.g. circulating B cells) from said immunised camelid;

c) amplifying regions of said cDNA or genomic DNA to obtain amplifiedgene segments, each gene segment comprising a sequence of nucleotidesencoding a VH domain or a sequence of nucleotides encoding a VL domainof a camelid conventional antibody;

d) cloning the gene segments obtained in c) into expression vectors,such that each expression vector contains a gene segment encoding a VHdomain and a gene segment encoding a VL domain and directs expression ofan antigen binding polypeptide comprising said VH domain and said VLdomain, thereby producing a library of expression vectors;e) screening antigen binding polypeptides encoded by the libraryobtained in step d) for immunoreactivity with said target antigen, andthereby selecting an expression vector encoding an antigen bindingpolypeptide immunoreactive with said target antigen;f) optionally performing a light chain shuffling step and/or a heavychain shuffling step to select an expression vector encoding apotency-optimised antigen binding polypeptide immunoreactive with saidtarget antigen;g) optionally subjecting the gene segment encoding the VH domain of thevector selected in step e) or step f) and/or the gene segment encodingthe VL domain of the vector selected in step e) or step f) to germliningand/or codon optimisation; andh) cloning the gene segment encoding the VH domain of the vectorselected in part e) or f) or the germlined and/or codon optimised VHgene segment produced in step g) and the gene segment encoding the VLdomain of the vector selected in part e) or f) or the germlined and/orcodon optimised VL gene segment produced in step g) into a furtherexpression vector, in operable linkage with a sequence of nucleotidesencoding one or more constant domains of a human antibody, therebyproducing an expression vector encoding a chimeric antigen bindingpolypeptide comprising the VH and VL domains fused to one or moreconstant domains of a human antibody.

The invention also extends to expression vectors prepared according tothe above-described processes, and to a method of producing an antigenbinding polypeptide immunoreactive with a target antigen, the methodcomprising steps of:

a) preparing expression vector encoding an antigen binding polypeptideimmunoreactive with a target antigen using the method described above;

b) introducing said expression vector into host cell or cell-freeexpression system under conditions which permit expression of theencoded antigen binding polypeptide; and

c) recovering the expressed antigen binding polypeptide.

In one embodiment, the latter process encompasses bulk production-scalemanufacture of the antigen-binding polypeptide of the invention,particularly bulk-scale manufacture of therapeutic antibodies intendedfor use as pharmaceutically active agents, by recombinant expression. Insuch embodiments, the expression vector prepared in step a) and the hostcell/expression system used in step b) are selected to be suitable forlarge-scale production of recombinant antibodies intended foradministration to human patients. The general characteristics ofsuitable vectors and expression systems for this purpose are well knownin the art.

The invention will be further understood with reference to the followingnon-limiting experimental examples.

Examples 1 to 9 illustrate the process of raising an antibody against anexample antigen denoted “cytokine x”, starting from immunization ofllamas. The same general protocol can be adapted for any target antigenin any camelid species, hence the precise identity of “cytokine x” isnot material. The process is also illustrated for preparation of Fabsbinding IL-1 Beta (Example 15 onwards).

Various publications are cited in the foregoing description andthroughout the following examples, each of which is incorporated byreference herein in its entirety.

General Protocol Example 1 Immunization of Llamas

Immunizations of llamas (Lama glama) and harvesting of peripheral bloodlymphocytes as well as the subsequent extraction of RNA andamplification of antibody gene fragments were performed as described byDe Haard and colleagues (De Haard et al., J. Bact. 187: 4531-4541(2005)). One llama was immunized intramuscularly with recombinant humanCytokine x using Freund's complete adjuvant or an appropriateanimal-friendly adjuvant Stimune (Cedi Diagnostics BV, The Netherlands).Cytokine x (recombinantly expressed in engineered human cell line) waspurchased. Prior to immunization the lyophilized cytokine x wasreconstituted in PBS (Dulbecco) at a concentration of 250 μg/ml. Thellama received 6 injections at weekly intervals, the first twoinjections with 100 μg of cytokine per injection, the four lastinjections with 50 μg for each boost. Four days after the lastimmunization a blood sample (PBL1) of 150 ml was collected from theanimal and serum was prepared. Ten days after the last immunization asecond blood sample (PBL2) of 150 ml was taken and serum was prepared.Peripheral blood lymphocytes (PBLs), as the genetic source of the llamaimmunoglobulins, were isolated from the blood sample using aFicoll-Paque gradient (Amersham Biosciences) yielding between 1 and5×10⁸ PBLs. The maximal diversity of antibodies is expected to be equalto the number of sampled B-lymphocytes, which is about 10% (between9.2-23.2% (De Genst et al., Dev. Comp. Immunol. 30:187-98 (2006)) of thenumber of PBLs (1−5×10⁷). The fraction of conventional antibodies inllama serum is up to 80% of the amount of total immunoglobulin, whichmight be extrapolated to a similar fraction of B-lymphocytes thatproduce the conventional antibodies. Therefore, the maximal diversity ofconventional antibodies in the 150 ml blood sample is calculated as0.8−4×10⁷ different molecules. Total RNA was isolated from PBLsaccording to the method of Chomczynski et al. Anal. Biochem. 162:156-159(1987)).

Example 2 Enrichment of Antigen Reactive B Cells by Panning or FACsSorting (Optional)

In order to reduce the complexity of the sampled B cell repertoireenabling the efficient cloning of the recombinatorial Fab phage displaylibrary, antigen reactive B cells were enriched by a FACS sorting(Weitkamp et al., J. Immunol. Meth. (2003) 275: 223-237) usingfluorescently labelled antigen and a mAb recognizing camelidconventional antibody specifically (as B cell marker) or by a panningprocedure on immobilized antigen (Lightwood et al., J. Immunol. Meth.316:133-143 (2006)).

PBLs from immunized animals were isolated via a density centrifugationon Ficoll-Paque as described above. Optionally co-purified red bloodcells were lysed by resuspending the PBL pellet in 20 ml of lysis buffer(8.29 g/L NH4Cl, 1.09 g/L KHCO3 and 37 mg/L EDTA) at room temperaturefollowed by centrifugation for 10 minutes at 200×g. Optional was alsothe depletion of monocytes by adhering these to the plastic surface ofTI50 culture flask. To achieve this cells were resuspended in 70 ml RPMI(Invitrogen) supplemented with 10% foetal calf serum, Glutamax, 25 mMHepes, penicillin-streptomycin (Invitrogen) and 0.38% sodium citrate andincubated for 2 hours at 37° C. and 5% CO2 in the flasks. Thesupernatant fraction containing the B selected was recovered and cellswere counted.

Bulk sorting in FACS of (living) B cells displaying target specificconventional antibodies was performed by simultaneous staining with thefluorescently labeled mAb specifically recognizing camelid conventionalantibodies and target antigen, labeled with yet another fluorescent dye.Between 1,000 and 100,000 antigen specific cells were sorted and usedfor RNA extraction by applying the protocol of Gough and colleagues(Gough, Anal. Biochem. 173:93-95 (1988)) or by using the TRIzol kit(Invitrogen). Total RNA was converted into random primed cDNA astemplate for the amplification of the antibody heavy and light chainvariable genes (see Example 3 and further).

Example 3 Amplification and Cloning of Variable Region Genes

Random primed cDNA was prepared from 80 μg of PBL RNA using thesuperscript III First-Strand Synthesis System for RT-PCR (Invitrogen).RNA was heat-denatured for 5 min at 65° C. in the presence of 2.5 μM ofrandom hexanucleotide primer and 500 μM dNTPs in 8 independent reactionof 20 ul reaction. Subsequently, buffer and dithiothreitol were addedaccording to the supplier's instructions, as well as 640 units ofRNasOUT (40 units/μl, Invitrogen), and 3200 units of SuperscriptIIIreverse transcriptase (200 units/μl; Invitrogen) in a total final volumeof 8×40 μl. After 50 min at 50° C., 5 min at 85° C. and 1 min at 1° C.RNAse H was added (˜4U) and incubated for 20 min at 37° C. The pooledcDNA was cleanup using QIAquick PCR Purification Kit according tosupplier's recommendation and used for PCR.

Primers annealing to the 3′ end of CH1 and 5′ and 3′ end of VH weredesigned on the basis of germline sequences from llama and dromedary,for which the deposited sequences could be retrieved from IMGT and otherdatabases following the citations from De Genst and colleagues (De Genstet al, Dev. Comp. Immunol. 30:187-198 (2006)). For design ofoligonucleotides for amplification of the light chain the rearranged andsomatically mutated dromedary sequences were used that were published ina thesis study (I. Legssyer, Free University Brussels).

All primary PCRs were carried out with separate BACK primers annealingto the 5′ end of the variable region and combined with FOR primersannealing to the 3′ end of CH1, on relatively large amounts of randomprimed cDNA as template (up to 2.5 μl corresponding to 6 μg of totalRNA) to maintain maximal diversity. The heavy chain derived ampliconscan be reamplified with a combination of JHFOR primers, annealing to the3′ end of VH and containing a naturally occurring BstEII site, andSfiI-tagged VHBACK primers annealing to the 5′ end of the VH gene, andsubsequently cloned as VH fragments. The light chain V-genes wereobtained by PCR with a set of CKFOR or CLFOR primer annealing to the 3′end of the constant domain and BACK primers priming at the 5′ end of theV-regions. The amplicons from the first PCR reactions were reamplifiedwith extended CH1FOR (containing a NotI site) or CKFOR and CLFOR primers(containing an AscI site) and subsequently cloned as llama Fabfragments. Alternatively, the DNA segments can be reamplified withprimers tagged with restriction sites (FOR primers with AscI site andFR4 based BACK primers with XhoI site) and cloned as VL fragments thuscreating chimeric Fab's containing llama derived V regions combined withhuman C regions.

PCR was performed in a volume of 50 μl reactions using Phusionpolymerase (Finnzymes) and 500 pM of each primer for 28 cycles (1 min at96° C., 1 min at 60° C., and 1 min at 72° C. All products were purifiedfrom agarose gel with the QIAex-II extraction kit (Qiagen). As input forreamplification to introduce restriction sites, 100-200 ng of purifiedDNA fragment was used as template in a 100-μl reaction volume. The largeamount of input, ensuring the maintenance of variability, was checked byanalysis of 4 μl of the “unamplified” PCR mixture on agarose gel.

Example 4 Construction of the Primary and Secondary Camelid FabRepertoires

For the construction of the primary heavy chain and the two primarylight chain repertoires, the PCR products, appended with restrictionsites, were gel-purified prior to digestion and the different VH, VK,and VL families combined into three groups. The VHCH1 fragments weredigested with SfiI and NotI and the VKCK and VLCL fragments weredigested with ApaLI and AscI, and cloned into the phagemid vector pCB3(similar to vector pCES1 with adapted multiple cloning site). Thedigested fragment (1 to 2 μg) were ligated to digested and purified PCB3(2 to 4 μg) using T4-DNA ligase (Fermentas) at room temperature forseveral hours and then at 37° C. for 1-2 hours. The desalted ligationmixture for light or heavy chain pools was used for electroporation ofthe E. coli strain TG1, to create the one-chain libraries.

Alternatively, the VH fragments, 1.5 μg in total, may be digested withSfiI and BstEII (present in the VH) and ligated in a 100-200-μl reactionmixture with 9 units of T4-DNA ligase at room temperature to 4 μg,gel-purified vector PCB4 (similar to vector PCB3, but with the pill genedeleted). In addition, the VH gene segments may be cloned via SfiI andBstEII and the VK/VL gene segments via ApaLI and XhoI, yielding thechimeric Fd and VKCK and VLCL.

The Fab library was obtained by cloning of light chain fragments,digested from plasmid DNA prepared from the light chain repertoires,into the plasmid collection containing the heavy chain repertoires.Plasmid DNA, isolated from at least 3×10⁹ bacteria of the VL library(the donor vector), was digested with ApaLI and AscI for cloning of thegel purified DNA fragments in the acceptor vector that already containedthe heavy chain libraries, thus creating a separate Fab library withkappa light chains and another library consisting of Fabs with a lambdalight chain with a size of 1-10×10⁹ clones. Similarly, the VLCL or VKCKfrom the single chain library can be extracted from agarose gel usingApaLI/AscI and cloned into the VHCH library vector using the samerestriction site.

Example 5 Selection of the Library

The rescue of phagemid particles with helper phage M13-KO7 or VCSM-13was performed on 2-liter scale, using representative numbers of bacteriafrom the library for inoculation, to ensure the presence of at least 10bacteria from each clone in the start inoculum. For selections, 10¹³colony-forming units were used with antigens immobilized in immunotubes(Maxisorp tubes, Nunc) or in 96 wells microtiterplates (Maxisorp, Nunc)or with soluble biotinylated antigens. The amount of the immobilizedantigens was reduced 10-100 fold during subsequent selection rounds,starting at 10 μg/ml at round 1. Antigens were biotinylated at a ratioof 3 to 10 molecules of NHS-Biotin (Pierce) per molecule of antigenaccording to the supplier's recommendations and tested for theirbioactivity in a bioassay. Unless stated otherwise, the antigens wereused for selection at concentrations of 1 to 10 nM during round 1 and 10pM to 1 nM during subsequent rounds.

Example 6 Screening for Antagonistic Cytokine x Specific Fab's

Soluble Fab was produced from individual clones as described in Marks etal. (Marks et al., J. Mol. Biol. 222:581-597 (1991)), but preferably asmonoclonal phage (Lee et al., Blood 108:3103-3111 (2006)) to boost thesensitivity. Culture supernatants containing soluble Fab or Fabdisplaying phage were tested in ELISA with directly coated antigen orcaptured via immobilized streptavidin. Recombinant human cytokine x andstreptavidin were coated at 10 μg/ml in 0.1 M NaHCO₃, pH 9.6, for 16 hat 4° C. Following 3 washes with PBS, 0.1% (v/v) Tween 20, biotinylatedantigen was added for 30 to 60 minutes at room temperature at aconcentration of 0.5 μg/ml. The plates were blocked during 30 min atroom temperature with 2% (w/v) semi-skim milk powder (Marvel) in PBS orwith 1% casein solution (in PBS). The culture supernatant was diluted 1-or 5-fold in 2% (w/v) Marvel/PBS and incubated 2 h; bound Fab wasdetected with anti-myc antibody 9E10 (5 μg/ml) recognizing themyc-peptide tag at the carboxyl terminus of the heavy Fd chain, andrabbit anti-mouse-HRP conjugate (Dako). Following the last incubation,staining was performed with tetramethylbenzidine and H₂O₂ as substrateand stopped by adding 0.5 volume of 1M H₂SO₄; the optical density wasmeasured at 450 nm. Clones giving a positive signal in ELISA (over 2times background), were analyzed by BstNI or HinfI fingerprinting of thePCR products obtained by amplification with the oligonucleotide primersM13-reverse and geneIII-forward (4) or of the separate Fd and VKCK orVLCL amplicons.

Screening for the Fab's capacity to interfere with binding of cytokine xto its receptor was performed in an appropriate receptor—ligand bindingELISA. For this, low amounts of biotinylated cytokine x were incubatedwith Fab in culture supernatant on cytokine x-Receptor coated plates andbound cytokine x was subsequently detected with streptavidin-HRPconjugate. Positive hits were sequenced and Fab purified for determiningtheir potency (IC50) in the in vitro receptor—ligand assay and to assesstheir affinity in BIAcore on immobilized cytokine x.

Large scale induction of soluble Fab fragments from individual cloneswas performed on a 50-ml or 250-ml scale in 2×TY containing 100 μg/mlcarbenicillin and 2% glucose. After growth at 37° C. to an OD600 of 0.9,the cells were pelleted (10 min at 2,934×g) and resuspended in 2×TY withcarbenicillin and 1 mM isopropyl-1-thio-D-galactopyranoside (IPTG).Alternative the De Bellis procedure (De Bellis and Schwartz, NAR (1990)18(5): 1311) was followed using 0.2% in stead of 2% glucose, thuspermitting the direct addition of IPTG to the medium of the late logphase cells. Bacteria were harvested after 3.5 h of growth at 30° C. bycentrifugation (as before); periplasmic fractions were prepared byresuspending the cell pellet in 1 ml of ice-cold PBS. After 2-16 h ofrotating head-over-head at 4° C., the spheroplasts were removed by twocentrifugation steps; after spinning during 10 min at 3,400×g, thesupernatant was clarified by an additional centrifugation step during 10min at 13,000×g in an Eppendorf centrifuge. The periplasmic fractionobtained was directly used in the different functional assays (targetbinding ELISA, in vitro receptor—ligand binding assays and Biacore).

For sequencing, plasmid DNA was prepared from 5-ml cultures grown at 30°C. in LB-medium, containing 100 μg/ml carbenicillin and 2% glucose,using the Qiagen Mini-kit (Qiagen) or on amplicons with vector primersM13-reverse and geneIII-forward, which anneal at the borders of the Fabinsert.

Example 7 Large Scale Production and Purification of Lead Fab's

Fab inserts from 3 to 6 different leads were recloned via ApaLI—NotI inan expression vector (coded pCB5) identical to pCB3 including thehexahistidine and C-MYC tags fused to the carboxyterminus of the Fd, butlacking the bacteriophage M13 gene3. In parallel the V regions wererecloned with the appropriate combination of restriction enzymes andsequentially cloned in gene3 deleted vectors containing human CH1 and CKor CL for the expression of chimeric Fabs. After fingerprint analysis,individual clones obtained after recloning were grown on 50-ml or 250-mlscale and periplasmic fractions were prepared as described above. Fabfragment was IMAC purified and the correctly formed Fab was furtherpurified via Size Exclusion Chromatography using a Superdex 75HR column(Amersham Pharmacia Biotech). Depending on the cell based assayendotoxin was removed by passage over an anion exchange column(Source30Q, GE Healthcare), which was sensitized overnight in 1 M NaOHand subsequently equilibrated in D-PBS. The yield was determined bymeasuring the optical density at 280 nm using a molar extinctioncoefficient of 13 for Fabs.

The purified Fab's were tested in the in vitro receptor—ligand bindingassay and Biacore to confirm the observations made with the Fab fragmentas produced by the gene3 containing pCB3 vector. Finally the potency ofthe Fab was determined in the bioassay.

Example 8

In this example the llama derived lead Fabs against cytokine x werehumanized by using a soft randomization procedure targeting a small setof framework residues and by monovalent display of the resulting Fablibrary on the surface of filamentous phage in order to identify highaffinity framework sequences via affinity based selection(US2003/0190317 A1, incorporated herein by reference). For instance, fordromedary derived germline VH (IGHV1S20) a small library was generatedfor positions 5 (Val, Leu), 55 (Gly, Ala), 83 (Ala, Ser), 95 (Lys, Arg),96 (Ser, Ala) and 101 (Met, Val) maintaining 70% of the wild typeresidues (IMGT numbering). Amino acids in the hypervariable loops couldbe addressed in a similar way.

Site-directed mutagenesis to correct PCR errors or to introduceadditional specific single codon changes was performed essentially asdescribed by Kunkel et al. Curr. Protoc. Mol. Biol. CH8U8.1 (2001) oralternatively Synthetic genes encoding the variants ordered fromGeneArt. The template for site-directed experiments was single-strandedDNA from a humanized Fab clone.

Site directed mutagenesis was also used to directly change a limitednumber of amino acid codons for humanization or modification purposes inthe DNA encoding the wild type or humanized Fabs.

After selection the individual leads were tested on affinity and potencyand the best leads were chosen for reformatting into human Fab/IgGs.

Example 9 Expression of Human Fab Fragments and Human MonoclonalAntibodies

Starting from the humanized VH and VL regions expression of humanizedFabs was performed as described in Rauchenberger et al. J. Biol. Chem.278:38194-204 (2003). For the expression of human monoclonal antibodiestwo separate expressions vectors, one for the light chain and one forthe heavy chain construct were constructed based on the pcDNA 3.1vector. The expression vector for the light chain contained either thehuman C kappa or human C lambda sequence downstream of a CMV promoter aswell as restriction sites allowing the cloning of the light chainconstruct as KPN1 BsmB1 fragments downstream of the CMV promoter and inframe with the light chain constant domain. The expression vector forthe heavy chain contained then human CH1-hinge-CH2-CH3 sequencedownstream of a CMV promoter as well as restriction sites allowing thecloning of the VH construct as KPN1 BsmB1 fragments downstream of theCMV promoter and in frame with the heavy chain constant domains.

The VL and VH fragments were cloned into the appropriate expressionvectors as KPN1 BsmB1 fragments containing the Kozak sequence followedby the mouse IgG kappa leader sequences in frame with the respective VLor VH sequence. These sequences were obtained by gene synthesis andoptimized for expression in mammalian cells (Geneart).

For full length IgG production VH and VL expression vectors constructswere co-transfected into mammalian cells (HEK-293 (transient) orCHO(stable)). Supernatant from transiently transfected cells or fromstably transfected cells was purified via protein A chromatography.

Monoclonal antibodies or Fabs were tested in receptor binding assays andin bioassays and the best leads were selected for further development.

Example 10 Camelid vs Human Homology Analysis

Methodology

Sequences of germline llama and dromedary J regions, which encode forFR4 were compared with human sequences and were completely identical (10out of 10 residues match); the only exception is IGHJ4, which containsGlutamine (instead of Leucine or Methionine) on position 6 in FR4(alignments shown below). For somatically mutated VLambda FR4 fromdromedary 9 out of 10 residues match (90% homology), since most of theavailable sequences have Histidine in stead of Lysine or Glutamate orGlutamine on position 6 (alignment shown below). Finally there is again90% identity (9 out of 10 residues) in FR4 of the set of six availablesomatically mutated dromedary VKappa sequences, because position 3residue Serine deviates from what is found in the human germline JKsegments, i.e. Glutamine, Proline and Glycine (alignment shown below).

For VH, VLambda and VKappa the analysis is supported by alignments (seebelow). Sequence alignment was done with closest human germlinesequence, having identical H1 and H2 canonical folds.

Residues which don't align, but which appear in another family member(or subclass) of the same germline are considered as homologous, basedon the assumption that it is feasible to mutate them back to saidclosest human germline sequence.

Canonical structures are compared using the following programs:www.bioinf.ora.uk/abs/chothia and www.bioc.unizhch/antibody/Sequences/GermlinesNBase_hVK. Residues of analyzed camelidantibodies (or from other species) which do not fit exactly thecanonical fold algorithm are checked for their appearance in thesequence of members of the matching human germline family with the samecombination of canonical folds, or mentioned residues allowing the foldsare checked for their appearance within the family of camelid antibodiesto which the analyzed antibody belongs.

Results are presented in the following sections:

10.1—Dromedary VH

10.2—Lama glama VH

10.3—VL 1-40

10.4—VL 2-18

10.5—VL 3-1

10.6—VL 3-12

10.7—VKappa 2-40

10.8—J(H) region comparisons of llama and human

10.9—Comparison of light chain J regions

10.10—Lama pacos VH germline homology

10.11—Lama glama derived VH homology analysis

10.12—Lama glama derived VL analysis

10.1 Dromedary VH (SEQ ID NOS: 1-20) IGHV         FR1-IMGT          CDR1-IMGT  gene          (1-26)            (27-38)  1       10        20         30.........|.........|...... ...|........ M99679, IGHV3-53EVQLVESGG.GLIQPGGSLRLSCAAS GFTVSSNY.... AF000603, IGHV1S1EVQLVESGG.GLVQPGGSLRLSCAAS GFTFSSYY.... AJ245151, IGHV1S2QVQLVESGG.GLVQPGGSLRLSCAAS GFTFSSYY.... AJ245152, IGHV1S3EVQLVESGG.GLVQPGGSLRLSCAAS GFTFSSYY.... AJ245153, IGHV1S4EVQLVESGG.GLVQPGGSLRLSCAAS GFTFSSYY.... AJ245154, IGHV1S5EVQLVESGG.GLVQPGGSLRLSCAAS GFTFSSYY.... AJ245155, IGHV1S6EVQLVESGG.GLVQPGGSLRLSCAAS GFTFSSYY.... AJ245157, IGHV1S7EVQLVESGG.GLVQPGGSLRLSCAAS GFTFSSYD.... AJ245158, IGHV1S8EVQLVESGG.GLVQPGGSLRLSCAAS GFTFSSYY.... AJ245159, IGHV1S9EVQLVESGG.GLVQPGGSLRLSCAAS GFTFSSYY.... AJ245160, IGHV1S10EVQLVESGG.GLVQPGGSLRLSCAAS GFTFSGYY.... AJ245164, IGHV1S11EVQLVESGG.GLVQPGGSLRLSCAAS GFTFSSYY.... AJ245165, IGHV1S12EVQLVESGG.GLVQPGGSLRLSCAAS GFTFSSYY.... AJ245167, IGHV1S13AVQLVESGG.GSVQAGGSLRLSCAAS GFTFSSYY.... AJ245168, IGHV1S14QVQLVESGG.GSVQAGGSLRLSCAAS GFTFSSYY.... AJ245170, IGHV1S15AVQLVESGG.GLVQPGGSLRLSCAAS GFTFSSYW.... AJ245171, IGHV1S16EVQLVESGG.GLVQPGGSLRLSCAAS GFTFSSYW.... AJ245173, IGHV1S17QVQLVESGG.GSVQAGGSLRLSCAAS GFTFSSYD.... AJ245174, IGHV1S18EVQLVESGG.GLVQPGGSLRLSCAAS GFTFSSYA.... AJ245156, IGHV1S19EVQLVESGG.GLVQPGGSLRLSCAAS GFTFSSYY.... IGHV    FR2-IMGT      CDR2-IMGT    FR3-IMGT gene    (39-55)        (56-65)     (66-104)40        50         60         70 .|.........|..... ....|..... ....|..... M99679, IGHV3-53MSWVRQAPGKGLEWVSV IYSGGST... YYADSVK.GR AF000603, IGHV1S1MSWVRQAPGKGLEWVSG IYSDGST... YYGDSVK.GR AJ245151, IGHV1S2MSWVRQAPGKGLEWVSG IYSDGST... YYGDSVK.GR AJ245152, IGHV1S3MSWARQAPGKGLEWVSG IYSDGST... YYGDSVK.GR AJ245153, IGHV1S4MSWVRQAPGKGLEWVSG IYSDGST... YYGDSVK.GR AJ245154, IGHV1S5MSWVRQAPGKGLEWVSG IYSDGST... YYGDSVK.GR AJ245155, IGHV1S6MSWVRQAPGKGLEWVSG IYSDGST... YYGDSVK.GR AJ245157, IGHV1S7MSWVRQAPGKGLEWVSG IYSDGST... YYGDSVK.GR AJ245158, IGHV1S8MSWVRKAQGKGLEWVSG IYSDGST... YYGDSVK.GR AJ245159, IGHV1S9MSWVRQAPGKGLEWVSS NTSDGST... YYGDSVK.GR AJ245160, IGHV1S10MSWVRQAPGRGLEWVSG IYSDGGT... YYGDSVR.GR AJ245164, IGHV1S11MSWVRQAPGKGLEWVSG IYSDGST... YYGDSVK.GR AJ245165, IGHV1S12MSWVRQAPGKGLEWVSG IYSDGST... YYGDSVK.GR AJ245167, IGHV1S13MSWVRQAPGKGLEWVSG IYSDGST... YYGDSVK.GR AJ245168, IGHV1S14MSWVRQAPGKGLEWVSG IYSDGST... YYGDSVK.GR AJ245170, IGHV1S15MYWVRQAPGKGLEWVSG IYSDGST... YYGDSVK.GR AJ245171, IGHV1S16MYWVRQAPGKGLEWVSG IYSDGST... YYGDSVK.GR AJ245173, IGHV1S17MSWVRQAPGKGLEWVSA IYSDGST... NYADSVK.GR AJ245174, IGHV1S18MSWVRQAPGKGLEWVSA INSRGST... HYADSMK.GR AJ245156, IGHV1S19MSWVRQAPGKGLEWVSG IYSDGST... YYGDSVK.GR ICHV      FR3-IMGT                 CDR3-IMGT gene      (66-104)                 (105-115)   80        90        100       110....|.........|.........|.... .....|..... M99679, IGHV3-53FTISRDNSKNTLYLQMNSLRAEDTAVYYC AR......... AF000603, IGHV1S1FTISRDNAKNMLYLQMNSLKPEDTAVYYC AG......... AJ245151, IGHV1S2FTISRDNAKNMLYLQMNSLKPEDTAMYYC AJ245152, IGHV1S3FTISRDNAKNMLYLQMNSLKPEDTAMYYC AJ245153, IGHV1S4FTISRDNAKNTLYLQMNSLKPEDTAMYYC AJ245154, IGHV1S5FTISRDNAKNMLYLQVNSLKPEDTAVYYC AJ245155, IGHV1S6FTISRDNAKHMLYLQMHSLKPEDTAMYYC AJ245157, IGHV1S7FTISRDKAKNMLYLQMNSLKPEDTAMYYC AJ245158, IGHV1S8FTISRDKAKNMLYLQMNSLKPEDTAMYYC AJ245159, IGHV1S9FTISRDNAKNMLYLQMNSLKPEDTAMYYC AJ245160, IGHV1S10FTISRDNAKNMLYLQMNSLKPEDTAMCYC AJ245164, IGHV1S11FTISRDKAKNMLYLQMNSLKPEDTSMYYC AJ245165, IGHV1S12FTISRDKAKNMLYLQMNSLKPEDTAMYYC AJ245167, IGHV1S13FTISRDKAKNMLYLQMNSLKPEDTAMYYC AJ245168, IGHV1S14FTISRDKAKNMLYLQMNSLKPEDTAMYYC AJ245170, IGHV1S15FTISRDKAKNMLYLQMNSLKPEDTAMYYC AJ245171, IGHV1S16FTISRDKAKNMLYLQMNSLKPEDTAMYYC AJ245173, IGHV1S17FTISRDNAKNTVYLQMNSLKPEDTAMYYC AJ245174, IGHV1S18FTISRDNAKNVLYLQMNSLKPEDTAMYYC AJ245156, IGHV1S19FTISQDNAKNTVYLQMNSLKPEDTAMYYC A) Sequence comparisonsSequences in comparison to human IGHV3-23 and other human germlines: 55 G/S/A can be considered more variable/beginning of CDR2  68 Galso human A exist in the same context (IGHV1S17/18) ->should be replacebale by A  83 Apresent in many other human VH3 class germlines  86 M/Valso human T exist in the same context -> should be replacebale by T 95 K also in human germlines 3-15/49/72/73  96 Palso in human germline 3-19 101 M not found in human IGHV3 classSequence homology: 26/26 (FR1) + 17/17 (FR2) + 38/39 (FR3) = 81/82 = 99%B) Canonical folds analysis CDR H1 and H21) Analysis of germline Dromedary VH sequences IGHV1S1 to IGHV1S19reveals a canonical fold 1 for CDR1 and fold 1 for CDR2, so identicalto the folds of human germline VH3-13 and VH3-53, confirming thedata published by Nguyen and colleagues (EMBO J (2000), 19(5),p 921-930). The analysis for dromedary IGHV1S1 and human IGHV3-53are shown below as examples using auto-generated SDR templates: IGHV1S1CDR H1 Class ? ! Similar to class 1/10A, but: !H94 (Chothia Numbering) is deleted. CDR H2 Class 1/9A [1 gig] IGHV3-53CDR H1 Class ? ! Similar to class 1, but: !H94 (Kabat Numbering) is deleted. CDR H2 Class 1 chothia:human [1 gig](SEQ ID NOS: 21-42) IGHV         FR1-IMGT           CDR1-IMGT gene         (1-26)              (27-38) 1       10        20         30.........|.........|...... ...|....... M99660, IGHV3-23EVQLLESGG.GLVQPGGSLRLSCAAS GFTFSSYA... AJ245177, IGHV1S20EVQLVESGG.GLVQPGGSLRLSCAAS GFTFSSYY... AJ245178, IGHV1S21AVQLVESGG.GLVQPGGSLRLSCAAS GFTFSSYY... AJ245183, IGHV1S22EVQLVESGG.GLVQPGGSLRLSCAAS GFTFSSYW... AJ245185, IGHV1S23EVQLVESGG.GLVQPGGSLRLSCAAS GFTFSSYW... AJ245186, IGHV1S24EVQLVESGG.GLVQPGGSLRLSCAAS GFTFSSYW... AJ245187, IGHV1S25EVQLVESGG.GLVQPGGSLRLSCAAS GFTFSSYW... AJ245189, IGHV1S26EVQLVESGG.GLVQPGGSLRLSCAAS GFTFSSYW... AJ245191, IGHV1S27EVQLVESGG.GLVQPGGSLRLSCAAS GFTFSSYD... AJ245192, IGHV1S28EVQLVESGG.GLVQPGGSLRLSCAAS GFTFSSYD... AJ245193, IGHV1S29EVQLVESGG.GLVQPGGSLRLSCAAS GFTFSSYA... AJ245194, IGHV1S30EVQLVESGG.GLVQPGGSLRLSCAAS GFTFSSYA... AJ245195, IGHV1S31AVQLVESGG.GLVQPGGSLRLSCAAS GFTFSSYA... AJ245179, IGHV1S32EVQLVESGG.GLVQPGGSLRLSCAAS GFTFSSYW... AJ245180, IGHV1S33EVQLVESGG.GLVQPGGSLRLSCAAS GFTFSSYW... AJ245182, IGHV1S34EVQLVESGG.GLVQPGGSLRLSCAAS GFTFSSYW... AJ245190, IGHV1S35EVQLVESGG.GLVQPGGSLRLSCAAS GFTFSSYW... AJ245196, IGHV1S36EVQLVESGG.GLVQPGGSLRLSCAAS GFTFSSYA... AJ245197, IGHV1S37EVQLVESGG.GSVQAGGSLRLSCAAS GFTFSSYD... AJ245181, IGHV1S38EVQLVESGG.GLVQPGGSLRLSCAAS GFTSSSYW... AJ245198, IGHV1S39EVQLVESGG.GLVQPGGSLRLSCAAS AFTYSSCC... AJ245199, IGHV1S40PEVQLVESGG.GLVQPGGSLRLSCAAS *FTFSSYA... IGHV    FR2-IMGT      CDR2-IMGT    FR3-IMGT gene    (39-55)        (56-65)     (66-104)40        50         60         70.|.........|..... ....|..... ....|..... M99660, IGHV3-23MSWVRQAPGKGLEWVSA ISGSGGST.. YYADSVK.GR AJ245177, IGHV1S20MSWVRQAPGKGLEWVSG INSDGSNT.. YYADSVK.GR AJ245178, IGHV1S21MSWVRQAPGKGLEWVSG IYTGGGST.. YYADSVK.GR AJ245183, IGHV1S22MYWVRQAPGKGLEWVST INSGGGST.. YYADSVK.GR AJ245185, IGHV1S23MYWVRQAPGKGLEWVST INSGGGST.. YYADSVK.GR AJ245186, IGHV1S24MYWVRQAPGKGPEWVST INSGGGST.. YYADSVK.GR AJ245187, IGHV1S25MYWVRQAPRKGLEWVST INSAGGST.. YYADSVK.GR AJ245189, IGHV1S26MYWVRQAPGKGLEWVST INSAGGST.. YYADSVK.GR AJ245191, IGHV1S27MSWVRQAPGKGLEWVSA INSGGGST.. YYADSVK.GR AJ245192, IGHV1S28MSWVRQAPGKGLEWVSA INSGGGST.. YYADSVK.GQ AJ245193, IGHV1S29ISWVRQAPGKGLEWVSA INSGGGST.. YYADSVK.GR AJ245194, IGHV1S30MSWVRQAPGKGLEWVSA INSGGGST.. YYADSVK.GR AJ245195, IGHV1S31ISWVRQAPGKGLEWVSA INSGGGST.. YYADSVK.GR AJ245179, IGHV1S32VYWVRQAPGKGLEWVSS IYTGGGST.. YYADSVK.GR AJ245180, IGHV1S33MYWVRQAPGKGLEWVSS IYTGGGST.. YYADSVK.GR AJ245182, IGHV1S34MSWVRQAPGKALQWVSS IYTGGGST.. YYADSVK.GP AJ245190, IGHV1S35MYWVRQAPGKGLEWVSA INSGGGST.. YYADSVK.GR AJ245196, IGHV1S36MSWVRQAPGKGLEWVSA IYTGGGST.. YYADSVK.GR AJ245197, IGHV1S37MSWVRQAPGKGLEWVSA INSGGGST.. YYADSVK.GR AJ245181, IGHV1S38MYWVRQAPGKGLEWVSS IYTGGGST.. YYADSVK.GR AJ245198, IGHV1S39MYWVRQAPGKGFEWVSA INSGGGST.. YYADSVK.GR AJ245199, IGHV1S40PISWVRQAPGKGLEWVSA INSGGGST.. YYADSVK.GR IGHV         FR3-IMGT              CDR3-IMGT gene         (66-104)              (105-115)   80        90        100       110....|.........|.........|.... .....|..... M99660, IGHV3-23FTISRDNSKNTLYLQMNSLRAEDTAVYYC AK......... AJ245177, IGHV1S20FTISRDNAKNTLYLQMNSLKSEDTAMYYC AJ245178, IGHV1S21FTISRDNAKNVLYLKLSSLKPEDTAMYYC AJ245183, IGHV1S22FTISRDNAKNMLYLQMNSLKPEDTAMYYC AJ245185, IGHV1S23FTISRDNAKNMLYLQMNSLKPEDTAVYYC AJ245186, IGHV1S24FTISRDNAKNMLYLQMNSLKPEDTAMYYC AJ245187, IGHV1S25FTISRDNAKNTVYLQMNSLKPEDTAMYYC AJ245189, IGHV1S26FTISRDNAKNTVYLQMNSLKPEDTAMYYC AJ245191, IGHV1S27FTISRDNAKNTLYLQMNSLKPEDTAMYYC AJ245192, IGHV1S28FTISRDNAKNTLYLQMNSLKPEDTAMYYC AJ245193, IGHV1S29FTISRDNAKNTVYLQLNSLKPEDTAMYYC AJ245194, IGHV1S30FTISRDNAKNTLYLQLNSLKTEDTAMYYC AJ245195, IGHV1S31FTISRDNAKNTVYLQLNSLKTEDTAMYYC AJ245179, IGHV1S32FTISKDNAKNTLYLQMNSLKPEDTAMYYC AJ245180, IGHV1S33FTISKDNAKNTLYLQMNSLKPEDTAMYYC AJ245182, IGHV1S34LTISKDNAKNTLYLQMNSLKPEDTAMYYC AJ245190, IGHV1S35FTISQDNAKNTRYLQMNSLKPEDTAMYYC AJ245196, IGHV1S36FTISQDNAKNTVYLQMNSLKTEDTAMYYC AJ245197, IGHV1S37FTISQDNAKNMLYLQMNSLKPEDTAMYYC AJ245181, IGHV1S38FTISKDNAKNTLYLQMNSLKPEDTAMYYC AJ245198, IGHV1S39FTISQDNAKNTRYLQMNSLKPEDTAMYYC AJ245199, IGHV1S40PFTISQDNAKNTRYLQMNSLKPEDTAMYYC A) Sequence comparisonsSequences in comparison to human IGHV3-23 and other human germlines:  5 V exist in all other VH3 germlines  55 G/T/Scan be considered more variable/beginning of CDR2  83 Apresent in many other human VH3 germlines  86 M/Vmajority is human T in the same context -> should be replaceable by T 95 K also in human germlines 3-15/49/72/73  96 Palso in human germline 3-19 101 M not found in human IGHV3 classSequence homology: 26/26 (FR1) + 17/17 (FR2) + 38/39 (FR3) = 81/82 = 99%B) Canonical folds analysis CDR H1 and H22) Analysis of the germline Dromedary VH sequences IGHV1S20 and IGH1S22to IGH1S24 shows the presence of canonical fold 1 for CDR1 and fold 3for CDR2, so identical to the canonical folds found in human germlineVH3-23, confirming the data published by Nguyen and colleagues (EMBO J(2000), 19(5), p 921-930). The analysis for dromedary IGHV1S20 and humanIGHV are shown below as examples, where it should be mentioned thatresidue H57 Asparagine is found back in VH3-21 that also have a class3 fold for CDR2 in combination with class 1 fold of CDR1 (deletions areD- and/or J-region encoded): IGHV1S20: CDR H1 Class ? !Similar to class 1/10A, but: ! H94 (Chothia Numbering) is deleted. !H102 (Chothia Numbering) = - (allows: YHVISDG) CDR H2 Class ? !Similar to class 3/10B, but: ! H52 (Chothia Numbering) =N (allows: SFWH) IGHV3-21 CDR H1 Class ? ! Similar to class 1, but: !H94 (Chothia Numbering) is deleted. CDR H2 Class 3 chothia:human [1igc]3) Germline Dromedary VH sequences IGH1S21 and IGHV1S25 to IGHV1S39have canonical fold 1 for CDR1 and fold 2 for CDR2, identical to thefolds of human germline VH5, thereby confirming the data published byNguyen and colleagues (EMBO J (2000), 19(5), p 921-930). The analysisfor IGHV1S32 is shown below as an example using auto-generated SDRtemplates. Serine (S) in position H55 is not found nor Lysine (K)in human germline VH with fold 2 of CDR2: IGHV1S32: CDR H1 Class ? !Similar to class 1/10A, but: ! H94 (Chothia Numbering) is deleted. !H102 (Chothia Numbering) = - (allows: YHVISDG) CDR H2 Class ? !Similar to class 2/10A, but: ! H50 (Chothia Numbering) =S (allows: REWYGQVLNKA) ! H71 (Chothia Numbering) = K (allows: VAL)Compared to results for IGHV3-23: CDR H1 Class ? !Similar to class 1/10A, but: ! H94 (Chothia Numbering) is deleted. !H102 (Chothia Numbering) = K (allows: YHVISDG) CDR H2 Class ? !Similar to class 2/10A, but: ! H71 (Chothia Numbering) = R (allows: VAL)Conclusions:Canonical fold 1 for CDR1-and fold 1 for CDR2 were predicted for alarge group of germline dromedary VH (similar as in human germlineVH3-13), another large group used canonical fold 1 for CDR1 and fold3 for CDR2 (as in human germline VH321) and another group usedcanonical fold 1 for CDR1 and fold 2 for CDR2 (as found in humangermline VH3-21) Note: Residues 50, 52, 71, 94, 102 according to Chothianumbering compare with residues 55, 57, 80, 107, 117 in IMGT numberingsystem, germline VH3-21) respectively.

10.2 Llama VH (SEQ ID NO: 21 AND 23) IGHV         FR1-IMGT           CDR1-IMGT      FR2-IMGT gene          (1-26)             (27-38)       (39-55)1       10        20         30        40        50.........|.........|...... ...|.........|.........|.....M99660, IGHV3-23EVQLLESGG.GLVQPGGSLRLSCAAS GFTFSSYA....MSWVRQAPGKGLEWVSAAF305949, IGHV1S6EVQLVESGG.GLVQPGGSLRLSCAAS GFTFSSSA....MSWVRQAPGKGLEWVSS IGHVCDR2-IMGT                  FR3-IMGT                 CDR3-IMGT gene (56-65)                   (66-104)                 (105-115)      60         70        80        90        100        110....|..... ....|.........|.........|.........|.... .....|.....M99660, IGHV3-23ISGSGGST.. YYADSVK.GRFTISRDNSKNTLYLQMNSLRAEDTAVYYC AK.........AF305949, IGHV1S6IYSYSSNT.. YYADSVK.SRFTISTDNAKNTLYLQMNSLKPEDTAVYYC AA.........A) Sequence comparisons:Sequences in comparison to human IGHV3-23 and other human germlines: 5 V exist in all other VH3 germlines 55 Sin 3-21/38; can be considered more variable/beginning of CDR2 74 Snot in huam gemline class 3 but in 4 and 6 80 Tnot present in any other human VH3 germlines 83 Ain many othe human class 3 germlines 95 Lnot present in any other human VH germlines 96 Pnot present in class 3 but in 2 and 6 Sequence homology: 26/26 (FR1) +17/17 (FR2) + 35/39 (FR3) = 78/82 = 95% B) Canonical folds analysis:M99660, IGHV3-23 Results using auto-generated SDR templates:CDR H1 Class ? ! Similar to class 1/10A, but: !H94 (Chothia Numbering) is deleted. ! H102 (Chothia Numbering) =K (allows: YHVISDG) CDR H2 Class ? ! Similar to class 2/10A, but: !H71 (Chothia Numbering) = R (allows: VAL)AF305949, IGHV1S6 Results using auto-generated SDR templates:CDR H1 Class ? ! Similar to class 1/10A, but: !H32 (Chothia Numbering) = S (allows: IHYFTNCED) !H94 (Chothia Numbering) is deleted. ! H102 (Chothia Numbering) =A (allows: YHVISDG) CDR H2 Class ? ! Similar to class 2/10A, but: !H50 (Chothia Numbering) = S (allows: REWYGQVLNKA) !H56 (Chothia Numbering) = N (allows: YREDGVSA) !H71 (Chothia Numbering) = T (allows: VAL)(B) Canonical folds CDR H1 and H2Analysis of the only available germline Llama VH sequence aligning to humangermline IGHV3-23 reveals a canonical fold 1 for CDR1 and fold 2 for CDR2identical to the folds of CDR1 and 2 of some human germline VH2 members andall VH5 and VH7 members. The analysis for IGHV1S6 is shown below:Discussion analysis:1) Position 32 Serine is also found in human germline VH2-8, VH3-20 andVH3-22, which all have a canonical fold 1 for CDR12) Position 94 is deleted, position 102 is A; these residues are encodedby the D and/or J region3) Position 50 Serine (S) for canonical fold 2 for CDR2 is not found inhuman germline VH with the same fold4) Position 56 Asparagine (N) is found in VH2-9 and VH2-10 that sharecanonical fold 2 for CDR25) Position 71 Threonine (T) is found in almost all human germline VHwith canonical fold 2 for CDR2 Conclusion:Llama germline sequence IGHV1S6 is most likely adopting canonical folds1/10A for CDR1 and 2/10A for CDR2 just as found for the human IGHV3-23germline sequence. Note: Chothia numbering 32, 50, 56, 71, 94 and 102compare to numbers 33, 55, 62, 77, 107 and 115 in IMGT numbering scheme,respectively.

10.3 VL 1-40 (SEQ ID NOS: 44 AND 45) IGLV         FR1-IMGT           CDR1-IMGT  gene          (1-26)             (27-38)1       10        20         30         40.........|.........|...... ...|........ .|.. M94116, IGLV1-40QSVLTQPPS.VSGAPGQRVTISCTGS SSNIGAGYD... VHWY Camv144QSVLTQPPS.MSGSLGQRVTISCTGS SSNIGGGSG... VQWF IGLVFR2-IMGT      CDR2-IMGT           FR3-IMGT gene(39-55)        (56-65)            (66-104)      50         60         70        80.......|..... ....|..... ....|.........|.... M94116, IGLV1-40QQLPGTAPKLLIY GNS....... NRPSGVP.DRFSGSK..SG Camv144QQLPGTAPKLLIY GNS....... NRASGIP.DRFSESK..SG IGLV  FR3-IMGT            CDR3-IMGT       gene  (66-104)            (105-115)      FR4     90       100        110.....|.........|.... .....|...... M94116, IGLV1-40TSASLAITGLQAEDEADYYC QSYDSSLSG... Camv144SSASLTITGLQADDEADYYC ASYDNRLSG--PVFGGGTKLTVLG A) Sequence comparisons:11 M: not found in any human sequence 14 S:not in human VL1 sequences but in almost all other VL types 15 L:not in human VL1 but in VL 3,4 9, 10 classes 40 Q:not in human VL1 but in VL6-57 42 F: in 3-27 and V17 but not in class 168 A: not in any human VL 78 E: not in any human VL 85 5:not in any human VL 90 T: not in human VL1 but in classes 2-4 and 6-897 D: not in VL1 but in 4-3 and 8-61 Sequence homology: 23/26 (FR1) +15/17 (FR2) + 34/39 (FR3) = 72/82 = 88% homologyB) Canonical folds analysis:M94116, IGLV1-40: Results using auto-generated SDR templatesCDR L1 Class ? ! Similar to class 6/14A, but: !L31 (Chothia Numbering) = Y (allows: H) ! L32 (Chothia Numbering) =D (allows: N) ! L93 (Chothia Numbering) = S (allows: R)CDR L2 Class 1/7A [1lmk]Camv144: Results using auto-generated SDR templates CDR L1 Class ? !Similar to class 6/14A, but: ! L31 (Chothia Numbering) = S (allows: H) !L32 (Chothia Numbering) = G (allows: N) ! L93 (Chothia Numbering) =N (allows: R) CDR L2 Class 1/7A [1lmk]CDR L2 gives a perfect match for canonical fold 1/7A, while for CDR L1 threekey residues are questionable for giving a perfect match with canonical fold6/14A, which now will be discussed individually.1) Position L31 Serine (S, in CDR1) and L32 Glycine (G, in CDR1) should beHistidine (H) and Asparagine (N), respectively; both residues were not foundin sequences with a canonical fold 14A; no other camelid lambda sequences,which align with human germline VL1 family, were identified, thereforemaking the analysis rather difficult2) Position L93 Asparagine (N, in CDR3) should be Arginine (R), butAsparagine (N) also occurs in two human germline VL4 family members, whichshare the canonical fold 14A with human germline VL1-2 Conclusions:1) Canonical fold 1/7(A) for CDR2 expected and probably fold number 6/14Afor CDR1, i.e. identical to what is found in human germline VL1 member 1-40.NOTE: Positions 31, 32 and 93 (Chothia) compare to 34, 35, and 109 inIMGT nomenclature used for the sequence comparison.

10.4 VL 2-18 (SEQ ID NOS: 46-59) IGLV                             FR1-IMGT     CDR1-IMGT geneleader                        (1-26)       (27-38)                    1       10        20         30                    .........|.........|...... ...|Z73642, IGLV2-18                                   QSALTQPPS.VSGSPGQSVTISCTGT SSDV Camv117MAWALLLLTLLTQGTGSWA QSALTQPSS.VSGTPGQTVTISCTGT SNDV Camv133MAWALLLLTLLTQGTGSWA QSALTQPSS.VSGTPGQTVTITCTGT RDDV Camv136MAWALLLLTLLTQGTGSWA QSALTQPSS.VSGTPGQTVTISCTGT SNDV Camv159MAWALLLLTLLTQGTGSWA QSALTQPSS.VSGTPGQTVTISCTGT SNDV Camv130MAWALLLLTLLTQGTGSWA QSALTQPSS.VSGTPGQTVTISCTGT SNDV Camv132MAWALLLLTLLTQGTGSWA QSALTQPSS.VSGTPGQTVTISCTGT SNGV Camv157MAWALLLLTLLTQGTGSWA QSALTQPSS.VSGTPGQTVTISCTGT SNDV Camv15MAWALLLLTLLTQGTGSWA QSALTQPSS.VSGTPGQTVTISCTGT SNDV Camv165                    QAVLTQPSS.VSGTPGQTVTISCTGT SNDV Camv151MAWALLLLTLLTQGTGSWA QSALTQPSS.VSGTPGQTVTISCTGT SNDV Camv131MAWALLLLTLLTQGTGSWA QSALAQPSS.VSGTPGQTVTISCTGT SNDV Camv160MAWALLLLTLLTQGTGSWA QSALTQPSS.VSGTPGQTVTISCTGT RDDV Camv152MAWALLLLTLLTQGTGSWA QSALTQPSS.VSGTPGQTVTISCTGT SNDV IGLVCDR1-IMGT   FR2-IMGT     CDR2-IMGT      FR3-IMGT gene(27-38)     (39-55)       (56-65)       (66-104)         40        50         60         70........ .|.........|..... ....|..... ....|........  Z73642, IGLV2-18GSYNR... VSWYQQPPGTAPKLMIY EVS....... NRPSGVP.DRFSG Camv117GGYNY... VSWYQQLPGTAPKLLIY QDS....... KRNSGIP.DRFSG Camv133GGYNY... VSWYQQLPGTAPKLLIY QIN....... KRLSGIP.DRFSG Camv136GGYNY... VSRYQQLPGTAPKLLIY QIN....... KRASGIP.DRFSG Camv159GRYNY... VSWYQQLPGTAPKFLIY QVN....... KRASGIP.DRFSG Camv130GRYNY... VSWYQQFPGTAPKLLIY QVN....... KRASGIP.DRFSG Camv132GGYNY... VSWYRQLPGTAPKLLIY QVN....... KRASGIP.DRFSG Camv157GGYNY... VSWYQQLPGTAPKLLIY QVN....... KRASGIP.DRFSS Camv15GRYNY... VSWYQQLPETAPKLLIY DVD....... KRASGIP.DRFSG Camv165GRYNY... VSWYQQFPGTAPKLLIY QVN....... KRGSG1P.DRFSG Camv151GGYNY... VSWHQQVPGTAPKLILY QVK....... ERPSGIP.DRFSG Camv131GRYNY... VSWYQQLPGTAPKLLIY QVN....... KRPSGIP.DRFSG Camv160GKYNY... VSWYQQLPGTAPKLLIY QVN....... KRASGIP.DRFSG Camv152GRYAY... VSWYQHLPGTAPKLLIY QVN....... KRASGTP.DRFSG IGLV   FR3-IMGT                 CDR3-IMGT gene   (66-104)                 (105-115)       FR480        90       100        110.|.........|.........|.... .....|......  Z73642, IGLV2-18SK..SGNTASLTISGLQAEDEADYYC SLYTSSSTF... Camv117SK..SDNTASMTISGLQSADEADYYC ASYRSTYH---SL FGGGTHLTVLG Camv133SK..SGNTASMTISGLQSADEADYYC ASYRDLNT---LV FGGGTHLTVLG Camv136SR..SGNTASMTISGLQSADEADYYC ASYRATNS---IV FGGGTHLTVLG Camv159SK..SGNTASMTISGLQSADEADYYC ASLPSSGN---AV FGGGTHLTVLG Camv130SK..SGNTASMTISGLQSADEADYYC ASYRNWAN---LP FGGGTHLTVLG Camv132SK..SGNTASMTISGLQSADEADYYC ASYRNGNN---AV FGGGTHLTVLG Camv157SK..SDNTASMTISGLQSADEADYYC ASYRSRDN---AV FGGGTHLTVLG Camv15SK..SGNTASMTISGLQSADEADYYC ASYRSGDN---AA FGGGTRLTVLG Camv165SK..SGNTASMTISGLQSADEADYYC ASLSSGNN---AV FGGGTHLTVLG Camv151SK..FGNTASMTISGLQSADEADYYC ASYSSPNN---VL FGGGTHLTVLG Camv131SK..SGNTVSLTISGLQSADEADYTC ASYKHTYN---AV FGGGTHLTVLG Camv160SK..SGNTASMTISGLQSADEADYYC ASVRDYDNNEFVV FGGGTHLTVLG Camv152SK..SGNTASMTTSGLQSADEADYYC SAYRSNDGP---V FGGGTHLTVLGA) Sequence comparisons:  8 Snot in class 2 but also in 3-27 and 4-60, 5-52 14 Tnot in class 2 but also in 1-44/47 18 Tnot in class 2 but in many other germlines of class 3 and in manyother germline classes as well 45 L not in class 2, only in class 1 53 Lin 2-33, as well as in classes 1, 4, 5, 7, 8, 10 66 K in classes 1, 2, 368 N/L/A/Gsomatic mutation and also human residue P is found in Camv151 andCamv131 71 Iin other class 2 members and in class 1-41/51 and classes 3, 4, 9, 1089 M somatic mutation (?) as also human residue L is fund in Camv13196 S in classes 1, 2, 4, 5 97 A not found in human germline.Sequence homology: 23/26 (FR1) + 16/17 (FR2) + 38/39 (FR3) = 78/82 =94% homology B) Canonical folds analysis:Z73642, IGLV2-18: Results using auto-generated SDR templatesCDR L1 Class ? ! Similar to class ?/14C, but: !L32 (Chothia Numbering) = R (allows: Y) ! L90 (Chothia Numbering) =L (allows: S) ! L93 (Chothia Numbering) = S (allows: G)CDR L2 Class 1/7A [1lmk]Camv159 Results using auto-generated SDR templates CDR L1 Class ? !Similar to class ?/14C, but: ! L28 (Chothia Numbering) = N (allows: S) !L93 (Chothia Numbering) = S (allows: G) CDR L2 Class 1/7A [1lmk]Analysis of the camelid lambda light chain variable sequences aligning to humangermline IGLV2-18 reveals a canonical fold 2/14 for CDR1 and fold 1/7A for CDR2,so identical to the folds of CDR1 and 2 of human germline VL2 family. Theanalysis for Camv159 is shown as an example:CDR L2 gives a perfect match for canonical fold 1/7A, while for CDR L1 only twokey residues are questionable for giving a perfect match with canonical fold2/14C, which now will be discussed individually.1) Position L28 Asparagine (N, in CDR1) should be Serine (S), but Aspartate (D)found in Camv133 is the most often found residue in human germline VL2 sequences2) Position L93 Serine (S, in CDR3) should be Glycine (G), but Serine presentin three of the 8 aligned camelid VLs is the most often found residue in humangermline VL2, as in the human IGLV2-18 used for comparison Conclusions:Canonical fold number 2/14 for CDR1 expected and fold 1/7(A) for CDR2, i.e.identical to what is found in human germline VL2. NOTE: Position 93(Chothia) compares to 109 in IMGT nomenclature used for the sequencecomparison.

10.5 VL 3-1 (SEQ ID NOS: 60-65) IGLV            FR1-IMGT           CDR1-IMGT  gene             (1-26)             (27-38) 1       10        20         30         40.........|.........|...... ...|..........|... X57826, IGLV3-1SYELTQPPS.VSVSPGQTASITCSGD KLGDKY...... ACWYQ Camv119QSVLTQPSA.VSVSLGETARITCQGG NFGSYY...... ANWYQ Camv120QSVLTQPSA.VSVPLGETARITCQGG DFGDYY...... VSWYQ Camv18 TALTQPSA.VSVSLGETARITCQGG NFGSYY...... TSWYQ Camv118QAVLSQPSA.VSVSLGETARITCQGD NFGSYY...... FSWYQ Camv123QSVLTQPSA.VSVSLGQTARITCQGG ILGSKK...... TNWYQ IGLV   FR2-IMGT    CDR2-IMGT       FR3-IMGT gene   (39-55)      (56-65)        (66-104)     50         60         70        80......|..... ....|..... ....|.........|...... X57826, IGLV3-1QKPGQSPVLVIY QDS....... KRPSGIP.ERFSGSN..SGNT Camv119QKPGQAPVLVLY KDS....... ARPSGIP ERFSGSS  SGGT Camv120QKPGQSPVLVIY KDT....... LRPSGIP ERFTGSS  SGGA Camv18QKPEEAPVVVIY KDT....... ERPSGIP ERFSASS  SGDT Camv118QKPGQAPVLVIY RNS....... NRPSGIP ERFSASS  SGGT Camv123QKPGQAPVLVIY GDD....... SRPSGIP ERFSGSR  SGGT IGLV  FR3-IMGT            CDR3-IMGT gene   (66-104)            (105-115)  90       100        110 ...|.........|.... .....|......X57826, IGLV3-1 ATLTISGTQAMDEADYYC QAWDSSTA.... Camv119ATLTISGAQAEDEADYYC QSGSSSA-SA--V FGGGTHLTVLG Camv120ATLTISGAQAEDEADYYC QSETSSA-T---V FGGGTHLTVLG Camv18ATLTISGAQAEDEADYYC QSGSSSA-NAP-V FGGGTKLTVLG Camv118ATLTISGAQAEDEADYYC QSADSSGRNAR-A FGGGTKLTVLG Camv123ATLTISGAQAEDEADYYC QLLDSTDSSSYWV FGGGTHLTVLG C) Sequence comparisons: 1 Q primer encoded  2 S/T primer encoded  3 V/A primer encoded  5 Sprimer encoded  8 S also in 3-27 and 4-60  9 A also in 3-19/32 15 Lin 3-9/16/19/32 17 Enot in human class 3 but human Q exists in Camv123 in same context 20 Rin all other human class 3 sequences and others 24 Q in 3-19/32 26 Gsomatic Mutation/part of CDR; human D exists in Camv118 in same context39 V/T/F somatic mutation/part of CDR 40 N/Ssomatic mutation/part of CDR 49 A in many other class 3 66 A/L/N/Ssomatic mutation/part of CDR 80 S in 3-16/19/10/25/27 85 Gnot in class 3 but in classes 6 and 7 94 A in 3-19/10/9 97 Ein 3-10/16/19 Sequence homology: 26/26 (FR1) + 17/17 (FR2) +38/39 (FR3) + 81/82 = 99% homology D) Canonical folds analysis:Analysis of the above listed camelid lambda light chain variable sequences on theUCL webpage http://www.bioinf.org.uk/abs/chothia.html shows that CDR-L1 adoptscanonical fold 11, while CDR-L2 has canonical fold 7. This corresponds with thecanonical folds found in all human germline IGLV3 family members (see Pluckthunhomepage http://www.bioc.unizh.ch/antibody/Sequences/Germlines/VBase_hVK.html). Thecanonical fold is determined by the length of the CDRs and certain key residueswithin and outside of the CDR. The analysis of e.g. Camv120 is shown below:CDR L1 Class ? ! Similar to class 2/11A, but: ! L2 (Chothia Numbering =S (allows: I) ! L25 (Chothia Numbering = G (allows: A) !L26 (Chothia Numbering = G (allows: S) ! L28 (Chothia Numbering =F (allows: NSDE) ! L29 (Chothia Numbering = G (allows: IV) !L51 (Chothia Numbering = D (allows: ATGV) ! L71 (Chothia Numbering =A (allows: YF) ! L90 (Chothia Numbering = S (allows: HQ)CDR L2 Class 1/7A [1lmk]CDR L2 gives a perfect match for canonical fold 1/7A, while for CDR L1 certain keyresidues are questionable for giving a perfect match with canonical fold 2/11A,which now will be discussed individually.1) Position L2 Serine (S) should be Isoleucine (I), but human IGLV3-5 (which alsohas fold 11) has Serine (S) residue found in these camelid VLs.2) L25 Glycine (G) is found in all human VL3 family members3) L26 Glycine (G) should be Serine (S), but Aspartate (D) found in Camv118 ismost often used in human VL3 members4) L28 Phenylalanine (F, in CDR1) should be Asparagine (N), Serine (S), Aspartate(D) or Glutamate (E), but Leucine(L) found in Camv123 is the most often usedresidue in human VL35) L29 Glycine (G, in CDR1) is found in 4 of the 9 human VL3 germlines6) L51 Aspartate (D, in CDR2) is most often used in human VL37) L71 Alanine (A, FR3) is most often used in human VL38) L90 Serine (S, CDR3) is most used residue in human VL3 Conclusions:2) Canonical fold number 11 for CDR1 is expected and fold 7 for CDR2, i.e.identical to what is found in human germline VL3 Note: Chothia Numbering51, 71, and 90 compares to IMGT numbering 57, 87, and 106

10.6 VL 3-12 (SEQ ID NOS: 66 and 67) IGLV                 FR1-IMGT          CDR1-IMGT geneleader            (1-26)            (27-38)        1       10        20         30         40        .........|.........|...... ...|........ .|... 273658, IGLV3-12        SYELTQPHS.VSVATAQMARITCGGN NIGSKA...... VHWYQ Camvlll        QSVLTQPST.ASMSLGQTAKITCQGG SLRNYA...... AHWYQ IGLV          FR2-IMGT    CDR2-IMGT          FR3-IMGT  geneleader    (39-55)      (56-65)           (66-104)             50         60         70        80        ......|..... ....|..... ....|.........|...... 273658, IGLV3-12        QKPGQDPVLVIY SDS....... NRPSGIP.ERFSGSN..PGNT Camvlll        QKPGAAPVLVIY NDN....... NRPSGIP.ERFSGSK..SGGT IGLV         FR3-IMGT      CDR3-IMGT geneleader   (66-104)      (105-115)             FR4          90       100        110                   ...|.........|.... .....|...... 273658, IGLV3-12        TTLTISRIEAGDEADYYC QVWDSSSDH... Camvlll        ATLTISRTKAEDEADYYC LSRDMSDSN-RVV FGGGTHLTVLGA) Sequence comparisons:  1 Q primer encoded  2 S primer encoded  3 Vprimer encoded  5 S primer encoded  8 S also in 3-27 and 4-60  9 Tnot in any human germline 11 Anot in class 3 but in classes 1, 2, 4, and 9 13 Mnot in any human germline 14 Sin many other germlines of class 3 and in many other germline classes as well15 L in 3-16/19/32 and in many other classes 16 Galmost everywhere other than in this particular germline 18 Tin many other germlines of class 3 and in many other germline classes as well20 K not in human class 3 sequences but in class 4 24 Q in 3-19/32 26 Gsomatic mutation/part of CDR 39 A in many other germlines of class 348 A not in any human germline 49 Ain many other germlines of class 3 and in many other germline classes as well80 K in 3-32 and many other germeline classes 85 Gnot in class 3 but in class 7 87 Ain many other germlines of class 3 and in many other germline classes as well94 T in 3-1 95 K not in class 3 but in 2-33 and:6-57 97 Ein 3-10/16/19 and in many other germline classesSequence homology: 22/26 (FR1) + 16/17 (FR2) + 37/39 (FR3) + 75/82 =91% homologyMost likely homology underestimated due to somatic mutations and because only onefamily member is found. B) Canonical folds analysis:Z73658, IGLV3-12: Results using auto-generated SDR templatesCDR L1 Class ? ! Similar to class 2/11A, but: ! L2 (Chothia Numbering) =Y (allows: I) ! L25 (Chothia Numbering) = G (allows: A) !L26 (Chothia Numbering) = N (allows: S) ! L28 (Chothia Numbering) =I (allows: NSDE) ! L29 (Chothia Numbering) = G (allows: IV) !L51 (Chothia Numbering) = D (allows: ATGV) ! L71 (Chothia Numbering) =T (allows: YF) ! L90 (Chothia Numbering) = V (allows: HQ)CDR L2 Class 1/7A [1lmk]Camvlll: Results using auto-generated SDR templates CDR L1 Class ? !Similar to class 2/11B, but: ! L25 (Chothia Numbering) = G (allows: A) !L26 (Chothia Numbering) = G (allows: N) ! L29 (Chothia Numbering) =R (allows: P) ! L34 (Chothia Numbering) = H (allows: Y) !L46 (Chothia Numbering) = L (allows: M) ! L71 (Chothia Numbering) =A (allows: V) ! L90 (Chothia Numbering) = S (allows: A) !L93 (Chothia Numbering) = M (allows: N) CDR L2 Class 1/7A [1lmk]Remarks: more difficult to align, especially FR1. These are somatically mutatedsequences, meaning that variations in FR can be expected as well. Deviations fromcanonical fold analyis are very comparable in IGLV3-12, so most likely adoptscomparable canonical folds. For CDR L2 fold 1-7A shows a perfect match, CDR L2will adopt fold 2-11 just as in human counterpart. Conclusion:Adopts canonical fold 2/11 for CDR1 and fold 1/7A for CDR2, so identical to humangermline VL3. Only one family member found, which is somatically mutated andtherefore it is difficult to draw conclusions.

10.7 VKappa 2-40 (SEQ ID NOS: 68-75) IGKV       FR1-IMGT             CDR1-IMGT      FR2-IMGT      CDR2-IMGT gene        (1-26)              (27-38)        (39-55)        (56-65)1       10        20         30         40        50         60.........|.........|...... ...|........ .|.........|..... ....|..X59314, IGKV2-40DIVMTQTPLSLPVTPGEPASISCRSS QSLLDSDDGNTY LDWYLQKPGQSPQLLIY TLS.... Kp6DIVMTQSPSSVTASVGEKVTINCKSS QSVFDTSRQKSF LNWHRQRPGQSPRRLIY YAS.... Kp48DIVMTQSPSSVTASVGEKVTINCKSS QSVFSSSSQKSL LDWHQQRPGQSPRRLIY YAS.... Kp3DIVMTQSPSSVTASVGEKVTINCKSS QHVISVSNQKSY LNWYQQRPGQSPRLLIY YAS.... Kp20DIVMTQSPSSVTASVGEKVTINCKSS QSVLSSSNQKSY LNWYQQRPGQSPRLLIY YAS.... Kp7DIVMTQSPSSVLASVGEKVTINCKSS QSVLSSSNQKSY LNWYQQRPGQSPRLLIT YAS.... Kp10DIVMTQSPTSVTASVGEKVTINCKSS QSVFASSSQKSQ LAWHQQRPGQSPRRLIY YAS.... Kp1DIVMTQSPSSVTASVGEKVTINCKSS QNLVSDSNQRSL LAWHQQRPGQSPRKLIY YAS.... IGKV                   FR3-IMGT                 CDR3-IMGT gene                   (66-104)                 (105-115)       70        80        90        100       110... ....|.........|.........|.........|.... .....|..... X59314, IGKV2-40... YRASGVP.DRFSGSG..SGTDFTLKISRVEAEDVGVYYC MQRIEFP.... Kp6... TRQSGVP.DRFSGSG..STTDFTLTISSVQPEDAAVYYC QQAFNVQPS FGSGTRLEIKR Kp48... ARASGVP.DRFSGSG..STTDFTLTISSVQPEDAAVYYC QQYSGSPPT FGSGTRLEIKR Kp3... TRESGIP.DRFSGSG..STTDFALTISSVQPEDAAVYYC QQAYSTPYS FGSGTRLEIKR Kp20... TRESGIP.DRFSGSG..STTDFTLTISSVQPEDAAVFYC QQAYSAPYS FGSGTRLEIKR Kp7... TRESGIP.DRFSGSG..STTDFTLTISSVQPEDAVVYYC QQAYSKPYN FGNGTRLEIKR Kp10... TRESGVP.DRFSGSG..STTDFTLTISSVQPEDAAVYYC QHLYSAPYS FGSGTRLEIKR Kp1... TRISGTP.DRFSGSG..STTDFTLTISSVQPEDAAVYYC QQGKKDPLS FGSGTRLEIKRA) Sequence comparisons:   7 S primer encoded   9 Sonly in human kappa 1 not kappa 2 family  11 Vonly in human kappa 1-12 not kappa 2 family  12 T/Lnot in human kappa germline  13 A not in class 2 but in class 1 and 5 14 S not in class 2 but in class 1, 3, 4, 7  15 V only in class 1  19 Vnot in class 2 but in class 1, 5, 6  20 Tnot in class 2 but in class 1, 3, 4, 6, 7  22 Nnot in class 2 but in class 4  24 K in classes 2, 4, 5  40 N/Asomatic mutation, human D also exists in KP48  42 Hsomatic mutation, human Y also exists in KP3/20/7  43 Q/R Q also found in classes 1, 2, 3, 4, 5, 6 and 7; R is somatic mutation 51 R in 2-24 and 2-30 as well as is class 3  52 R/KR in 2-30 and 1-17; human L also exits  66 T/Ain classes 1, 3, 4 but not in 2; somatic mutation/part of CDR  68 Q/Esomatic mutation, human A also exists in KP48  71 Isomatic mutation; human V also exists in same context in various dromedarykappa chains  84 T not in any human kappa germline  90 Tnot in class 2 but in classes 1, 3, 4, 5, 6, 7  93 Snot in class 2 but in classes 1, 3, 4, 6  95 Qnot in class 2 but in classes 1, 3, 4  96 Pnot in class 2 but in classes 1, 3  99 Anot in class 2 but in classes 5, 6 100 A/Vnot in class 2 but in classes 1, 3, 4, 5, 6, 7Sequence homology: 17/26 (FR1) + 17/17 (FR2) + 31/39 (FR3) = 66/82 =80% homologyMost likely homology underestimated due to somatic mutations and because all sequences seem to belong to the same class. B) Canonical folds analysis:X59314, IGKV2-40 Results using auto-generated SDR templates CDR L1 Class ? ! Similar to class 3/17A, but: !L90 (Chothia Numbering) = Q (allows: N) CDR L2 Class 1/7A [1lmk]Kp6 Results uing auto-generated SDR templates CDR L1 Class ? !Similar to class 3/17A, but:  ! L29 (Chothia Numbering) = V (allows: L)! L90 (Chothia Numbering) = Q (allows: N) CDR L2 Class 1/7 [1lmk]Analysis of the camelid kappa light chain variable sequences aligning to human germlineIGLV2-40 reveals a canonical fold 3/17A for CDR1 and fold 1/7A for CDR2, so identical tothe folds of CDR1 and 2 of human germline IGKV2-40. Dromedary Kp6 was analyzed as anexample. CDR2 gives a perfect match for canonical fold 1, while for CDR1 a number ofresidues are questionable for giving a perfect match with canonical fold 3/17A, whichnow will be discussed individually.1) Positiion L29 Valine (V, in CDR1) should be Leucine (L) for having canonical fold 3for CDR1, but this residue is present in dromedary Kp1 sequence2) Position L90 Glutamine (Q, in CDR3) occurs as well in human germline VBase_VK4_1,which also has canonical folds 3 for CDR1 (in combination with fold 1 for CDR2)Conclusions: Canonical fold 1 for CDR2 expected and most probably foldnumber 3/17A for CDR1 and fold 1 for CDR2, i.e. identical to what isfound in human germline NOTE: Postitions 90 (Chothia) compares toposition 107 in IMGT nomeclature used for the sequence comparison.

10.8 J(H) region comparisons of human SEQ ID NOS: 76-81) and llama (SEQ ID NOS: 82-86) Human IGHJ Genes---CDR3--- ----FR4----  1        10        20  ......... |.........|J00256, IGHJ1  ...AEYFQH WGQGTLVTVSS J00256, IGHJ2 ...YWYFDL WGRGTLVTVSS J00256, IGHJ3  .....AFDV WGQGTMVTVSSJ00256, IGHJ4  .....YFDY WGQGTLVTVSS J00256, IGHJ5 ....NWFDS WGQGTLVTVSS J00256, IGHJ6  YYYYYGMDV WGQGTTVTVSS Llama: IGHJGenes --CDR3--- ----FR4----   1        10   ....... ..|........AF305952, IGHJ2   GYRYLEV WGQGTLVTVSS AF305952, IGHJ3    NALDA WGQGTLVTVSS AF305952, IGHJ4      EYDY WGQGTQVTVSSAF305952, IGHJ5     PQFEY WGQGTLVTVS AF305952, IGHJ6 (1)     DFGS WGQGTLVTVS Sequence analysis:The J(H) regions derived from llama show a perfecthomology to their human counterparts. There is a100% sequence identity. The only exception is the6^(th) residue of FR4 in llama IGHJ4 being Gln (Q).This residue is not found in human J(H) regions at this position.

10.9 Comparison of lambda (SEQ ID NOS: 87-96)and kappa (SEQ ID NOS: 97-103) light chain J regions Lambda J:Kabat numbering ..97 98......108                       ---FR4----          -- CDR3 --  -J-lambda-                   ..  ..........X04457, IGLJ1     YV  FGTGTKVTVL M15641, IGLJ2     VV  FGGGTKLTVLM15642, IGLJ3     VV  FGGGTKLTVL X51755, IGLJ4     FV  FGGGTQLIILX51755, IGLJ5     WV  FGEGTELTVL M18338, IGLJ6     NV  FGSGTKVTVLX51755, IGLJ7     AV  FGGGTQLTVLCamv119 etc.      ..  FGGGTHLTVLG (38/44 anaylzed sequences)Camv18/18/4       ..  FGGGTKLTVLG (3/44 analyzed sequences)Camv158/28/5      ..  FGGGTRLTVLG (3/44 analyzed sequences)Sequence analysis:The first 2 residues of the human germline J are considered part of the CDR3.They are often changed during the joining process. The best match of dromedaryJ-lambda regions is with IGLJ2 and 3. 9/10 amino acids are identical: 90%sequence identity. Only His or Arg in position 103 (Kabat) do not match. Argis positively charged (as Lys) and even His can be considered somewhat positivelycharged. Therefore exchange to the human Lys should be possible.Kappa J: Kabat numbering ..97 98......108                      ---FR4----           -- CDR3 --    Kappa                  ..  .......... J00242, IGKJ1     WT  FGQGTKVEIKJ00242, IGKJ2     YT  FGQGTKLEIK J00242, IGKJ3     FT  FGPGTKVDIKJ00242, IGKJ4     LT  FGGGTKVEIK J00242, IGKJ5     IT  FGQGTRLEIKKp6/48/3/20/10/1      FGSGTRLEIKR (6/7 analyzed sequences)Kp7                   FGSGTRLEIKR (1/7 analyzed sequences)Sequence analysis:The first 2 residues of the human germline J regions are considered part of theCDR3. They are often changed during the joining process. Best match of dromedaryJ-kappa is with human IGKJ5. 9/10 amino acids match: 90% sequence identity.

10.10 Lama pacos VH germline analysis (SEQ ID NOS: 269-325) % %                       FR1 Name Ident Homol 1  2  3  4  5  6  7  8  9 10 11 12 13 14 15 X92343|IGHV1-46*01 Q  V  Q  L  V  Q  S  G  A  E  V  K  K  P  G LpVH1-s6 (AM939701) 89.2%91.9%  E  V  Q  L  V  Q  P  G  A  E  L  R  N  P  G LpVH1-s2 (AM939697)89.2% 91.9%  E  V  Q  L  V  Q  P  G  A  E  L  R  N  P  GLpVH1-s3 (AM939698) 87.8% 90.5% E  V  Q  L  V  Q  P  G  A  E  L  R  N  P  G LpVH1-s4 (AM939699) 89.2%91.9%  E  V  Q  L  V  Q  P  G  A  E  L  R  N  P  GLpVH1-s5 Ps (AM939700) 89.2% 91.9% E  V  Q  L  V  Q  P  G  A  E  L  R  N  P  G M99660|IGHV3-23*01 E  V  Q  L  L  E  S  G  G  G  L  V  Q  P  G AM939712 94.6% 98.6% E  V  Q  V  V  E  S  G  G  G  L  V  Q  P  G AM939713 89.2% 94.6% Q  L  Q  L  V  E  S  G  G  G  L  V  Q  P  G AM939730 93.2% 97.3% E  V  Q  L  V  E  S  G  G  G  L  V  Q  P  G AM939731 91.9% 97.3% E  V  Q  L  V  E  S  G  G  G  L  V  Q  P  G AM939744 86.5% 93.2% Q  V  Q  L  V  E  S  G  G  G  L  V  Q  P  G AM939726 93.2% 97.3% Q  L  Q  L  V  E  S  G  G  G  L  V  Q  P  G AM939727 93.2% 97.3% Q  L  Q  L  V  E  S  G  G  G  L  V  Q  P  G AM939739 94.6% 97.3% E  V  Q  L  V  E  S  G  G  G  L  V  Q  P  G AM939740 94.6% 98.6% Q  L  Q  L  V  E  S  G  G  G  L  V  Q  P  G AM939741 94.6% 98.6% E  V  Q  L  V  E  S  G  G  G  L  V  Q  P  G AM939742 90.5% 95.9% Q  V  Q  L  V  E  S  G  G  G  L  V  Q  P  G AM939743 93.2% 97.3% E  V  Q  L  V  E  S  G  G  G  L  V  Q  P  G    FR1                                        CDR1 Name 16 17 18 19  20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35   x92343|IGHV1-46*01A   S  V  K    V  S  C  K  A  S  G  Y  T  F  T  

   LpVH1-s6 (AM939701) A   S  V  K V  S  C  K  A  S  G  Y  T  F  T  S  Y  Y  I  D LpVH1-s2 (AM939697)A   S  V  K  V  S  C  K  A  S  G  Y  T  F  T  S  Y  Y  I  DLpVH1-s3 (AM939698) A   S  L  K V  S  C  K  A  S  G  Y  T  F  T  S  Y  Y  I  D LpVH1-s4 (AM939699)A   S  L  K  V  S  C  K  A  S  G  Y  T  F  T  S  Y  Y  I  DLpVH1-s5 Ps (AM939700) A   S  V  K V  S  C  K  A  S  G  Y  T  F  T  S  Y  Y  I  D M99660|IGHV3-23*01G   S  L  R  L  S  C  A  A  S  G  F  T  F  S  

AM939712 G   S  L  R  L  S  C  A  A  S  G  F  T  F  S  D  Y  A  M  S  AM939713 G   S  L  R  L  S  C  A  A  S  G  F  T  F  D  D  Y  G  M  S  AM939730 G   S  L  R  L  S  C  A  A  S  G  F  T  F  S  S  Y  A  M  S  AM939731 G   S  L  R  L  S  C  A  A  S  G  F  T  F  G  S  Y  D  M  S  AM939744 G   S  L  K  H  S  C  A  A  S  G  L  T  F  G  S  Y  D  M  S  AM939726 G   S  L  R  V  S  C  A  A  S  G  F  T  F  S  S  Y  Y  M  S AM939727 G   S  L  R  V  S  C  A  A  S  G  F  T  F  S  S  Y  Y  M  S AM939739 G   S  L  R  L  S  C  A  A  S  G  F  T  F  S  S  Y  D  M  S  AM939740 G   S  L  R  L  S  C  A  A  S  G  F  T  F  S  S  Y  A  M  S  AM939741 G   S  L  R  L  S  C  A  A  S  G  F  T  F  S  S  Y  A  M  S  AM939742 G   S  L  R  L  S  C  A  A  S  G  F  T  F  S  S  Y  A  M  S  AM939743 G   S  L  R  L  S  C  A  A  S  G  F  T  F  S  S  Y  A  M  S     CDR1                           FR2 Name 35a 35b 35c 36 37 38 39 40 41 42 43 44 45 46 47 48 49 X92343|IGHV1-46*01  W  V  R  Q  A  P  G  Q  G  L  E  W  M  G LpVH1-s6 (AM939701)  W  V  R  Q  A  P  G  Q  G  L  E  W  M  G LpVH1-s2 (AM939697)  W  V  R  Q  A  P  G  Q  G  L  E  W  M  G LpVH1-s3 (AM939698)  W  V  R  Q  A  P  G  Q  G  L  E  W  M  G LpVH1-s4 (AM939699)  W  V  R  Q  A  P  G  Q  G  L  E  W  M  G LpVH1-s5 PS (AM939700)  W  V  *  Q  A  P  G  Q  G  L  E  W  M  G M99660|IGHV3-23*01  W  V  R  Q  A  P  G  K  G  L  E  W  V  S AM939712  W  V  R  Q  A  P  G  K  G  L  E  W  V  S AM939713  W  V  R  H  S  P  G  K  G  L  E  W  V  S AM939730  W  V  R  Q  A  P  G  K  G  P  E  W  V  S AM939731  W  V  R  Q  A  P  G  K  G  P  E  W  V  S AM939744  W  V  R  Q  A  P  G  K  G  P  E  W  V  S AM939726  W  V  R  Q  A  P  G  K  G  L  E  W  V  S AM939727  W  V  R  Q  A  P  G  K  G  L  E  W  V  S AM939739  W  V  R  Q  A  P  G  K  G  L  E  W  V  S AM939740  W  V  R  Q  A  P  G  K  G  L  E  W  V  S AM939741  W  V  R  Q  A  P  G  K  G  L  E  W  V  S AM939742  W  V  R  H  S  P  G  K  G  L  E  W  V  S AM939743  W  V  R  Q  A  P  G  K  G  L  E  W  V  S % %                               CDR2 Name Ident Homol 50 51 52 52a 52b 52C 53 54 55 56 57 58 59 60 61 62 63 64 65  X92343|IGHV1-46*01

                 

LpVH1-s6 ( 89.2% 91.9% R  I  D  P            E  D  G  G  T  K  Y  A  Q  K  F  Q  G LpvH1-S2 (89.2% 91.9%  R  I  D  P            E  D  G  G  T  K  Y  A  Q  K  F  Q  GLpVH1-s3 ( 87.8% 90.5% R  I  D  P            E  D  G  G  T  K  Y  A  Q  K  F  Q  G LpVH1-s4 (89.2% 91.9%  R  I  D  P            E  D  G  G  T  K  Y  A  Q  K  F  Q  GLpVH1-s5 Ps 89.2% 91.9% R  I  D  P            E  D  G  G  T  N  Y  A  Q  K  F  Q  GM99660|IGHV3-23*01

              

AM939712 94.6% 98.6%A  I  S  W             N  G  G  S  T  Y  Y  A  E  S  M  K  G AM93971389.2% 94.6% A  I  S  W             N  G  G  S  T  Y  Y  A  E  S  M  K  GAM939730 93.2% 97.3%A  I  N  S             G  G  G  S  T  Y  Y  A  D  S  V  K  G AM93973191.9% 97.3% A  I  N  S             G  G  G  S  T  Y  Y  A  D  S  V  K  GAM939744 86.5% 93.2%A  I  N  S             G  G  G  S  T  Y  Y  A  D  S  V  K  G AM93972693.2% 97.3% A  I  N  T             G  G  G  S  T  Y  Y  A  D  S  V  K  GAM939727 93.2% 97.3%T  I  N  T             G  G  G  S  T  Y  Y  A  D  S  V  K  G AM93973994.6% 97.3% A  I  N  S             G  G  G  S  T  Y  Y  A  D  S  V  K  GAM939740 94.6% 98.6%A  I  N  S             G  G  G  S  T  S  Y  A  D  S  V  K  G AM93974194.6% 98.6% A  I  N  S             G  G  G  S  T  S  Y  A  D  S  V  K  GAM939742 90.5% 95.9%A  I  N  S             G  G  G  S  T  S  Y  A  D  S  M  K  G AM93974393.2% 97.3% A  I  N  G             G  D  G  S  T  S  Y  A  D  S  V  K  G                        FR3 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 82a 82bX92343|IGHV1-46*01  R  V  T  MT  R  D  T  S  T  S  T  V  Y  M  E  L  S   S LpVH1-s6 (  R  V  T  F T  A  D  T  S  T  S  T  A  Y  V  E  L  S   S Lpvh1-S2 (  R  V  T  FT  A  D  T  S  T  S  T  A  Y  V  E  L  S   S LpVH1-s3 (  R  V  T  F  T  A  D  T  S  T  R  T  A  Y  V  E  L  S   S LpVH1-s4 (  R  V  T  FT  A  D  T  S  T  S  T  A  Y  V  E  L  S   S LpVH1-s5 Ps  R  V  T  FT  A  D  T  S  T  S  T  A  Y  V  E  L  S   S M99660|IGHV3-23*01 R  F  T  I S  R  D  N  S  K  N  T  L  Y  L  Q  M  N   S AM939712 R  F  T  I S  R  D  N  A  K  N  T  L  Y  L  Q  M  N   S AM939713 R  F  T  I   S  R  D  N  A  K  N  T  V  Y  L  Q  M  N   S AM939730 R  F  T  I  S  R  D  N  A  K  N  T  L  Y  L  Q  M  N   S AM939731 R  F  T  I S  R  D  N  A  K  N  T  L  Y  L  Q  M  N   S AM939744 R  F  T  I S  R  D  N  A  K  N  T  L  Y  L  Q  M  N   S AM939726 R  F  T  I S  R  D  N  A  K  N  T  L  Y  L  Q  M  N   S AM939727     R  F  T  I S  R  D  N  A  K  N  T  L  Y  L  Q  M  N   S AM939739 R  F  T  I S  R  D  N  A  K  N  T  V  Y  L  Q  M  N   S AM939740 R  F  T  I S  R  D  N  A  K  N  T  L  Y  L  Q  M  N   S AM939741 R  F  T  I S  R  D  N  A  K  N  T  L  Y  L  Q  M  N   S AM939742 Q  F  T  I S  R  D  N  A  K  N  T  L  Y  L  Q  M  N   S AM939743 R  S  T  I   S  R  D  N  A  K  N  T  L  Y  L  Q  M  N   S AM939730 R  F  T  I  S  R  D  N  A  K  N  T  L  Y  L  Q  M  N   S AM939731 R  F  T  I S  R  D  N  A  K  N  T  L  Y  L  Q  M  N   S AM939744 R  F  T  I S  R  D  N  A  K  N  T  L  Y  L  Q  M  N   S AM939726 R  F  T  I S  R  D  N  A  K  N  T  L  Y  L  Q  M  N   S AM939727 R  F  T  I S  R  D  N  A  K  N  T  L  Y  L  Q  M  N   S AM939739 R  F  T  I S  R  D  N  A  K  N  T  V  Y  L  Q  M  N   S AM939740 R  F  T  I S  R  D  N  A  K  N  T  L  Y  L  Q  M  N   S AM939741 R  F  T  I S  R  D  N  A  K  N  T  L  Y  L  Q  M  N   S AM939742 Q  F  T  I S  R  D  N  A  K  N  T  L  Y  L  Q  M  N   S AM939743 R  S  T  I   S  R  D  N  A  K  N  T  L  Y  L  Q  M  N   S               FR3 Name 82c 83 84 85 86 87 88 89 90 91 92X92343|IGHV1-46*01  L  R  S  E  D  T  A  V  Y  Y  C LpVH1-s6 ( L  R  S  E  G  T  A  V  Y  Y  C Lpvh1-S2 ( L  R  S  E  G  T  P  V  Y  Y  C LpVH1-s3 ( L  R  S  E  G  T  A  V  Y  Y  C LpVH1-s4 ( L  R  S  E  G  T  A  V  Y  Y  C LpVH1-s5 Ps L  R  S  E  G  T  A  V  Y  Y  C M99660|IGHV3-23*01 L  R  A  E  D  T  A  V  Y  Y  C AM939712 L  K  S  E  G  T  A  V  Y  Y  C AM939713 L  K  P  E  G  T  A  V  Y  Y  C AM939730 L  K  P  E  G  T  A  V  Y  Y  C AM939731 L  K  P  E  G  T  A  V  Y  Y  C AM939744 L  K  P  E  G  T  A  V  Y  Y  C AM939726 L  K  P  E  G  T  A  V  Y  Y  C AM939727 L  K  P  E  G  T  A  V  Y  Y  C AM939739 L  K  P  E  D  T  A  V  Y  Y  C AM939740 L  K  P  E  G  T  A  V  Y  Y  C AM939741 L  K  P  E  G  T  A  V  Y  Y  C AM939742 L  K  P  E  G  T  A  V  Y  Y  C AM939743 L  K  P  E  G  T  A  V  Y  Y  C % %                      FR1 Name IdentHomol  1  2  3  4  5  6  7  8  9 10 11 12 13 14 15 U29841|IGHV3-23*03 E  V  Q  L  L  E  S  G  G  G  L  V  Q  P  G AM939716 94.6% 97.3% E  V  Q  L  V  E  S  G  G  G  L  V  Q  P  G AM939728 90.5% 94.6% E  V  R  L  V  E  S  G  G  G  L  V  Q  P  G AM939738 91.9% 95.9% Q  V  Q  L  V  E  S  V  G  G  L  V  Q  D  G AM939710 89.2% 93.2% Q  V  Q  L  V  E  S  G  G  G  L  V  Q  A  G AM939748 93.2% 97.3% Q  V  Q  L  V  E  S  G  G  G  L  V  Q  P  G AM939750 94.6% 98.6% Q  V  Q  L  V  E  T  G  G  G  L  V  Q  P  G AM939751 91.9% 95.9% Q  L  Q  L  V  E  S  G  G  G  L  V  Q  P  G AM939767 90.5% 94.6% Q  V  Q  L  V  E  S  G  G  G  L  V  Q  P  G AM939768 93.2% 97.3% E  V  Q  L  V  E  S  G  G  G  L  V  Q  P  G AM939707 93.2% 97.3% E  V  Q  L  V  E  S  G  G  G  L  V  Q  P  G AM939708 93.2% 97.3% Q  V  Q  L  V  E  S  G  G  G  L  V  Q  P  G AM939709 91.9% 95.9% E  V  Q  L  V  E  S  G  G  G  L  V  Q  P  G AM939732 93.2% 97.3% Q  V  Q  L  V  E  S  G  G  G  L  V  Q  P  G AM939733 89.2% 94.6% Q  V  Q  L  V  E  S  G  G  G  L  V  Q  P  G AM939717 90.5% 95.9% Q  V  Q  L  V  E  S  G  G  G  L  V  Q  P  G AM939734 94.6% 97.3% E  V  Q  V  V  E  S  G  G  G  L  V  Q  P  G AM939735 91.9% 97.3% E  V  Q  L  V  E  S  G  G  G  L  V  Q  P  G AM939736 93.2% 97.3% Q  V  Q  L  V  E  S  G  G  G  L  V  Q  P  G AM939737 94.6% 98.6% Q  V  Q  L  V  E  S  G  G  G  L  V  Q  P  G    FR1                                           CDR1 Name  16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35      U29481|IGHV3-23*03  G  S  L  R   L  S  C  A  A  S  G  F  T  F  S 

  AM939716   G  S  L  R   L  S  C  A  A  S  G  F  T  F  S  S  S  A  M  SAM939728   G  S  L  R   L  S  C  A  A  S  G  F  T  F  S  S  S  A  M  SAM939738   G  S  L  R     L  S  C  A  A  S  G  R  T  F  S  S  S  A  M  SAM939710   G  S  L  R   L  S  C  A  A  S  G  L  T  F  S  S  Y  A  M  SAM939748   G  S  L  R   L  S  C  A  A  S  G  L  T  F  S  S  Y  Y  M  SAM939750   G  S  L  R   L  S  C  A  A  S  G  F  T  F  S  S  S  A  M  SAM939751   G  S  L  R   L  S  C  A  A  S  G  F  T  F  S  S  Y  A  M  GAM939767   G  S  L  R   L  S  C  A  A  S  G  F  T  F  S  S  Y  A  M  GAM939768   G  S  L  R   L  S  C  A  A  S  G  F  T  F  S  S  Y  D  M  SAM939707   G  S  L  R   L  S  C  A  A  S  G  F  T  F  S  S  Y  A  M  SAM939708   G  S  L  R   L  S  C  A  A  S  G  F  T  F  S  S  Y  A  M  SAM939709   G  S  L  R   L  S  C  A  A  S  G  L  T  F  S  S  Y  Y  M  SAM939732   G  S  L  R   L  S  C  A  A  S  G  F  T  F  S  S  Y  D  M  SAM939733   G  S  L  R   L  S  C  A  A  S  G  F  T  L  G  S  Y  D  M  SAM939717   G  S  L  R   L  S  C  A  A  S  G  F  T  F  G  S  Y  D  M  SAM939734   G  S  L  R    L  S  C  A  A  S  G  F  T  F  S  S  Y  A  M  SAM939735   G  S  L  R   L  S  C  A  A  S  G  F  T  F  D  N  Y  A  M  SAM939736   G  S  L  R   L  S  C  A  A  S  G  F  T  F  S  S  Y  A  M  SAM939737   G  S  L  R   L  S  C  A  A  S  G  F  T  F  S  S  Y  A  M  S   CDR1                      FR2 Name 35a 35b 35c 36 37 38 39 40 41 42 43 44 45 46 47 48 49 U29481|IGHV3-23*03  W  V  R  Q  A  P  G  K  G  L  E  W  V  S AM939716  W  V  R  Q  A  P  G  K  G  L  E  W  V  S AM939728  R  V  R  Q  V  P  G  K  G  L  E  W  V  S AM939738  W  V  R  Q  A  P  G  K  G  L  E  W  V  S AM939710  W  V  R  Q  A  P  G  K  G  L  E  S  V  S AM939748  W  V  R  Q  A  P  G  K  G  L  E  W  V  S AM939750  W  V  R  Q  A  P  G  K  G  L  E  W  V  S AM939751  W  A  R  Q  V  P  G  K  G  L  E  W  V  S AM939767  W  A  R  Q  V  P  G  K  G  L  E  W  V  S AM939768  W  V  R  Q  A  P  G  K  G  L  E  W  V  S AM939707  W  V  R  Q  A  P  G  K  G  L  E  S  V  S AM939708  W  V  R  Q  A  P  G  K  G  L  E  S  V  S AM939709  W  V  R  Q  A  P  G  K  G  L  E  S  V  S AM939732  W  V  R  Q  A  P  G  K  G  P  E  W  V  S AM939733  W  V  R  Q  A  P  G  K  G  P  E  W  V  S AM939717  W  V  R  R  A  P  G  K  G  L  E  W  V  S AM939734  W  V  R  Q  A  P  G  K  G  L  E  W  V  S AM939735  W  V  R  Q  A  P  G  K  G  L  E  W  V  S AM939736  W  V  R  R  A  P  G  K  G  L  E  W  V  S AM939737  W  V  R  Q  A  P  G  K  G  L  E  W  V  S % %                               CDR2 Name Ident Homol 50 51 52 52a 52b 52c 53 54 55 56 57 58 59 60 61 62 63 64 65U29481|IGHV3-23*03   

              

AM939716 94.6% 97.3%  S  I  Y  S           Y  S  S  N  T  Y  Y  A  D  S  V  K  G AM93972890.5% 94.6%   S  I  Y  S           Y  S  S  N  T  Y  Y  A  D  S  V  K  GAM939738 91.9% 95.9%  S  I  Y  S           Y  S  S  N  T  Y  N  A  D  S  V  K  G AM93971089.2% 93.2%   T  I  N  S           D  G  S  N  T  Y  Y  A  D  S  V  K  GAM939748 93.2% 97.3%  G  I  Y  S           D  G  S  D  T  Y  Y  A  D  S  V  E  G AM93975094.6% 98.6%   G  I  Y  S           D  G  S  D  T  Y  Y  A  D  S  V  K  GAM939751 91.9% 95.9%   G  I  Y  S           D  G  S  — T  Y  Y  A  D  S  V  K  G AM939767 90.5% 94.6%  G  I  Y  S           D  G  S  —  T  Y  Y  A  D  S  V  K  G AM93976893.2% 97.3%   G  I  Y  S           D  G  S  —  T  Y  Y  A  D  S  V  K  GAM939707 93.2% 97.3%  S  I  Y  S           Y  S  S  N  T  Y  Y  A  D  S  V  K  G AM93970893.2% 97.3%   S  I  Y  S           Y  S  S  N  T  Y  Y  A  D  S  V  K  GAM939709 91.9% 95.9%  T  I  Y  S           Y  G  G  N  T  Y  Y  A  D  S  V  K  G AM93973293.2% 97.3%   D  I  N  S           G  G  G  S  T  Y  Y  A  D  S  V  K  GAM939733 89.2% 94.6%  C  I  N  S           G  G  G  S  T  Y  Y  A  D  S  V  K  G AM93971790.5% 95.9%   Y  I  N  S           G  G  G  S  T  Y  Y  A  D  S  V  K  GAM939737 94.6% 97.3%  Y  I  N  S           G  G  G  S  T  Y  Y  A  D  S  V  K  G AM93973591.9% 97.3%   Y  I  N  S           G  G  G  S  T  Y  Y  A  D  S  V  K  GAM939736 93.2% 97.3%  Y  I  N  S           G  G  G  S  T  Y  Y  A  D  S  V  K  G AM93973794.6% 98.6%   D  I  N  S           G  G  G  S  T  Y  Y  A  D  S  V  K  G                          FR3 Name  66 67 68 69 70 71 72 72 74 75 76 77 78 79 80 81 82  U29481|IGHV3-23*03   R  F  T  I  S  R  D  N  S  K  N  T  L  Y  L  Q  M AM939716   R  F  T  I  S  T  D  N  A  K  N  T  L  Y  L  Q  M AM939728   R  F  T  I  S  T  D  N  A  K  N  T  L  Y  L  Q  M AM939738   R  F  T  I  S  R  D  N  A  K  N  T  L  Y  L  Q  M AM939710   R  F  T  I  S  R  D  N  A  K  N  T  L  Y  L  Q  M AM939748   R  F  T  I  S  R  D  N  A  K  N  T  L  Y  L  Q  M AM939750   R  F  T  I  S  R  D  N  A  K  N  T  L  Y  L  Q  M AM939751   R  F  T  I  S  R  D  N  A  K  N  T  L  Y  L  Q  M AM939767   R  F  T  I  S  R  D  N  A  K  N  T  V  Y  L  Q  M AM939768   R  F  T  I  S  R  D  N  A  K  N  T  V  Y  L  Q  M AM939707   R  F  T  I  S  R  D  N  A  K  N  T  L  Y  L  Q  M AM939708   R  F  T  I  S  R  D  N  A  K  N  T  L  Y  L  Q  M AM939709   R  F  T  I  S  R  D  N  A  K  N  T  L  Y  L  Q  M AM939732   R  F  T  I  S  R  D  N  A  K  N  T  L  Y  L  Q  M AM939733   R  F  T  I  S  R  D  N  A  K  N  T  V  Y  L  Q  M AM939717   R  F  T  I  S  R  D  N  A  K  N  T  V  Y  L  Q  M AM939734   R  F  T  I  S  R  D  N  A  K  N  T  V  Y  L  Q  M AM939735   R  F  T  I  S  R  D  N  A  K  N  T  V  Y  L  Q  M AM939736   R  F  T  I  S  R  D  N  A  K  N  T  L  Y  L  Q  M AM939737   R  F  T  I  S  R  D  N  A  K  N  T  L  Y  L  Q  M                         FR3 Name82a   82b   82c 83 84 85 86 87 88 89 90 91 92 U29481|IGHV3-23*03  N     S   L  R  A  E  D  T  A  V  Y  Y  C AM939716   N     S  L  K  S  E  D  T  A  V  Y  Y  C AM939728   N     S  L  K  S  E  G  T  A  V  Y  Y  C AM939738   N     S  L  K  P  E  G  T  A  V  Y  Y  C AM939710   N     S  L  K  P  D  G  T  A  V  Y  Y  C AM939748   N     S  L  K  P  E  G  T  A  V  Y  Y  C AM939750   N     S  L  K  P  E  G  T  A  V  Y  Y  C AM939751   N     S  L  K  S  E  G  T  A  V  Y  Y  C AM939767   N     S  L  K  P  E  G  T  A  V  Y  Y  C AM939768   N     S  L  K  P  E  G  T  A  V  Y  Y  C AM939707   N     S  L  K  S  E  D  M  A  V  Y  Y  C AM939708   N     S  L  K  S  E  G  T  A  V  Y  Y  C AM939709   N     S  L  K  P  E  G  T  A  V  Y  Y  C AM939732   N     S  L  K  P  E  G  T  A  V  Y  Y  C AM939733   N     S  L  K  P  E  G  T  A  V  Y  Y  C AM939717   N     S  L  K  P  E  G  T  A  V  Y  Y  C AM939734   N     S  L  K  P  E  D  T  A  V  Y  Y  C AM939735   N     S  L  K  P  E  G  T  A  V  Y  Y  C AM939736   N     S  L  K  P  E  G  T  A  V  Y  Y  C AM939737   N     S  L  K  P  E  G  T  A  V  Y  Y  C % %                       FR1 NameIdent Homol   1  2  3  4  5  6  7  8  9 10 11 12 13 14 15 16L33851|IGHV-74*01   E  V  Q  L  v  E  S  G  G  G  L  V  Q  P  G  GAM939749 94.6% 98.6%   Q  V  Q  L  V  E  S  G  G  G  L  V  Q  P  G  GAM939724 93.2% 98.6%   Q  V  Q  L  V  E  S  G  G  G  L  V  Q  P  G  GAM939725 89.2% 95.9%   Q  L  Q  L  V  E  S  G  G  G  L  V  Q  P  G  GAM939745 87.8% 93.2%   E  V  Q  L  V  E  S  G  G  G  L  V  Q  P  G  GAM939723 93.2% 97.3%   Q  L  Q  L  V  E  S  G  G  G  L  V  Q  P  G  G     FR1                                      CDR1 Name  17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 35a L33851|IGHV-74*01  S  L  R  L   S  C  A  A  S  G  F  T  F  S  

AM939749   S  L  R  L   S  C  A  A  S  G  F  T  F  S  S  Y  W  M  NAM939724   S  L  R  L   S  C  A  A  S  G  F  T  F  S  S  Y  W  M  NAM939725   S  L  R  L   S  C  A  A  S  G  F  T  F  G  S  Y  W  M  YAM939745   S  L  R  L    S  C  A  A  S  G  F  T  F  G  S  Y  V  L  SAM939723   S  L  R  L   S  C  A  A  S  G  F  T  F  S  S  Y  W  M  N   CDR1                     FR2 Name 35b  35c 36 37 38 39 40 41 42 43 44 45 46 47 48 49 L33851|IGHV-74*01  W  V  R  Q  A  P  G  K  G  L  V  W  V  S AM939749  W  V  R  Q  A  P  G  K  G  L  E  W  V  S AM939724  W  V  R  Q  A  P  G  K  G  L  E  W  V  S AM939725  W  V  R  Q  A  P  G  K  G  L  E  W  V  S AM939745  W  V  C  H  S  P  G  K  G  L  E  W  V  S AM939723  W  V  R  Q  A  P  G  K  G  L  E  W  V  S % %                       FR1 Name Ident Homol  1  2  3  4  5  6  7  8  9 10 11 12 13 14 15 16 X92229|IGHV4-30-2*03  Q  L  Q  L  Q  E  S  G  S  G  L  V  K  P  S  Q LpVH2-s7 (AM939704)78.4% 81.1%   Q  V  Q  L  Q  E  S  G  P  G  L  V  K  P  S  Q                  FR1                                  CDR1 Name 17 18 19 20  21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 35a 35bX92229|IGHV4-30-2*03   T  L  S  L   T  C  A  V  S  G  G  S  I  S  

LpVH2-s7 (AM939704)   T  L  S  L    T  C  A  V  Y  G  G  S  I  T  T  S  C  Y  A  W  S CDR1                   FR2 Name 35c    36 37 38 39 40 41 42 43 44 45 46 47 48 49 X92229|IGHV4-30-2*03         W  I  R  Q  P  P  G  K  G  L  E  W  I  G LpVH2-s7 (AM939704)         C  I  C  Q  P  P  E  K  G  L  E  W  M  A % %                       FR1Name Ident Homol   1  2  3  4  5  6  7  8  9 10 11 12 13 14 15 16Z14238|IGHV4-30-4*01   Q  V  Q  L  Q  E  S  G  P  G  L  V  K  P  S  QLpVH2-s2 (AM939769) 86.5% 86.5%  E  V  Q  L  Q  E  S  G  P  G  L  V  K  P  S  Q LpVH2-s3 (AM939770)82.4% 82.4%   E  V  Q  V  Q  E  S  G  P  G  L  V  K  P  S  QLpVH2-s4 (AM939771) 82.4% 82.4%  Q  V  Q  R  Q  E  S  G  P  G  L  V  K  P  S  Q LpVH2-s5 (AM939772)87.8% 87.8%   Q  V  Q  L  Q  E  S  G  P  G  L  V  K  P  S  QLpVH2-s6 (AM939773) 82.4% 86.5%  E  V  Q  L  Q  E  S  G  P  G  L  L  K  P  S  Q LpVH20s11 Ps(AM939703)82.4% 82.4%   Q  V  Q  L  Q  E  S  G  P  D  L  V  K  P  S  QLpVH2-s8 (AM939705) 82.4% 82.4%  Q  V  Q  L  Q  E  S  G  P  G  L  V  K  P  S  Q LpVH2-s9 Ps(AM939706)83.8% 83.8%   Q  V  Q  L  *  E  S  G  P  G  L  V  K  P  S  QLpVH2-s10 (AM939702) 83.8% 83.8%  E  V  Q  L  Q  E  S  G  P  G  L  V  K  P  S  Q                  FR1                                  CDR1 Name 17 18 19 20  21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 35a 35bZ14238|IGHV4-30-4*01   T  L  S  L   T  C  T  V  S  G  G  S  I  S  

LpVH2-s2 (AM939769)   T  L  S  L  T  C  T  V  S  G  G  S  I  T  T  S  Y  Y  A  W  S LpVH2-s3 (AM939770)  A  L  S  L   T  C  T  A  S  G  G  S  N  T  T  S  Y  Y  A  W  SLpVH2-s4 (AM939771)   M  L  S  L    T  C  T  A  S  G  G  S  N  T  T  S  Y  Y  A  W  S LpVH2-s5 (AM939772)  T  L  S  L   T  C  T  V  S  G  G  S  I  T  T  S  Y  Y  Y  W  SLpVH2-s6 (AM939773)   T  L  S  L  T  C  A  V  Y  G  G  S  I  T  T  S  Y  Y  Y  W  SLpVH20s11 Ps(AM939703)   M  L  S  L  T  C  T  V  S  G  G  S  N  T  T  S  Y  Y  A  W  S LpVH2-s8 (AM939705)  T  L  S  L   T  C  T  A  S  G  G  S  N  T  T  S  Y  Y  A  W  SLpVH2-s9 Ps(AM939706)   T  L  S  L  T  C  T  V  S  G  G  S  I  T  T  S  C  Y  A  W  S LpVH2-s10 (AM939702)  M  L  S  L   T  C  T  L  S  G  D  S  I  T  T  S  C  Y  A  W  SCDR1                          FR2 Name 35c    36 37 38 39 40 41 42 43 44 45 46 47 48 49  Z14238|IGHV4-30-4*01         W   I  R  Q  P  P  G  K  G  L  E  W  I  G LpVH2-s2 (AM939769)         W  I  R  Q  P  P  G  K  G  L  E  W  M  G LpVH2-s3 (AM939770)         W    I  R  Q  P  P  G  K  G  L  E  W  M  G LpVH2-s4 (AM939771)         W    I  R  Q  P  P  G  K  G  L  E  W  M  G LpVH2-s5 (AM939772)         W  I  R  Q  P  P  G  K  G  L  E  W  M  G LpVH2-s6 (AM939773)           W  I  R  Q  P  P  G  K  G  L  E  W  M  G LpVH20s11 Ps(AM939703)         W  I  R  Q  P  P  G  K  G  L  E  *  M  G LpVH2-s8 (AM939705)         W  I  R  Q  P  P  G  K  G  L  E  W  M  G LpVH2-s9 Ps(AM939706)         W   I  H  Q  P  P  G  K  G  L  E  *  M  G LpVH2-s10 (AM939702)         W  I  R  Q  P  P  G  K  G  L  E  W  M  G % %                               CDR2 Name Ident Homol50 51 52 52a 52b 52c 53 54 55 56 57 58 59 60 61 62 63 64 65L33851|IGHV3-74*01   

              

  AM939749 94.6% 98.6%  G  I  Y  S           D  G  S  D  T  Y  Y  A  D  S  V  K G AM93972493.2% 98.6%   A  I  N  S           G  G  G  S  T  S  Y  A  D  S  M  K GAM939725 89.2% 95.9%  A  I  N  S           G  G  G  S  T  S  Y  A  D  S  M  K G AM93974587.8% 93.2%   A  I  N  S           C  G  G  S  T  S  Y  A  D  S  V  K GAM939723 93.2% 97.3%  A  I  N  S           G  G  G  S  T  Y  Y  A  D  S  V  K G                                                                              FR3 Name 66    67           68 69 70 71 72 73 74 75 76 77 78 79 80L33851|IGHV3-74*01   R     F   T  I  S  R  D  N  A  K  N  T  L  Y  LAM939749   R     F   T  I  S  R  D  N  A  K  N  T  L  Y  L AM939724  Q     F   T  I  S  R  D  N  A  K  N  T  L  Y  L AM939725   Q     F  T  I  S  S  D  N  A  R  N  T  L  Y  L AM939745   R     F  T  I  S  R  D  N  A  K  N  T  V  Y  L AM939723   R     F  T  I  S  R  D  N  A  K  N  T  V  Y  L                              FR3Name  81    82 82a 82b 83c 83 84 85 86 87 88 89 90 91 92L33851|IGHV3-74*01   Q     M   N  S   L   R  A  E  D  T  A  V  Y  Y  CAM939749   Q     M   N  S   L   K  P  E  G  T  A  V  Y  Y  C AM939724  Q     M   N  S   L   K  P  E  G  T  A  V  Y  Y  C AM939725   Q     M  N  S   L   K  P  E  G  T  A  V  Y  Y  C AM939745   Q     M  N  S   L   K  P  E  G  T  A  V  Y  Y  C AM939723   Q     M  N  S   L   K  P  E  G  T  A  V  Y  Y  C % %                               CDR2 Name Ident Homol50 51 52 52a 52b 52c 53 54 55 56 57 58 59 60 61 62 63 64 65X92229|IGHV4-30-2*03   

              

    LpVH2-s7 (AM939704) 78.4% 81.1%   A  I  —            Y  S  G  S  T  Y  Y  S  P  S  L  K  S                            FR3 Name  66    67 68 69 70 71 72 73 74 75 76 77 78 79 80 X92229|IGHV4-30-2*03   R     V  T  I  S  V  D  T  S  K  N  Q  F  S  L LpVH2-s7 (AM939704)   H     T  S  I  S  R  D  T  S  K  N  Q  F  S  L                              FR3Name  81    82 82a 82b 82c 83 84 85 86 87 88 89 90 91 92X92229|IGHV4-30-2*03   K     L   S  S   V   T  A  A  D  T  A  V  Y  Y  CLpVH2-s7 (AM939704)   Q     L   S  S   V   T  P  E  G  T  A  V  Y  Y  C% %                                CDR2 Name Ident Homol50 51 52 52a 52b 52c 53 54 55 56 57 58 59 60 61 62 63 64 64Z14238|IGHV4-30-4*01   

              

LpVH2-s2 (AM939769) 86.5% 86.5%  A  I  A             Y  S  G  S  T  Y  Y  S  P  S  L  K  SLpVH2-s3 (AM939770) 82.4% 82.4%  A  I  A             Y  S  G  S  T  Y  Y  S  P  S  L  K  SLpVH2-s4 (AM939771) 82.4% 82.4%  A  I  A             Y  D  G  S  T  Y  Y  S  P  S  L  K  SLpVH2-s5 (AM939772) 87.8% 87.8%  A  I  A             Y  D  G  S  T  Y  Y  S  P  S  L  K  SLpVH2-s6 (AM939773) 82.4% 86.5%  A  I  A             Y  D  G  S  T  Y  Y  S  P  S  L  K  SLpVH2-s11 Ps(AM939703) 82.4% 82.4%   A  I  —            Y  S  G  S  T  Y  Y  S  P  S  L  K  S LpVH2-s8 (AM939705)82.4% 82.4%   A  I  —             Y  S  G  S  T  Y  Y  S  P  S  L  K  SLpVH2-s9 Ps(AM939706) 83.8% 83.8%   A  I  —            Y  S  G  S  T  Y  Y  S  P  S  L  K  S LpVH2-s10 (AM939702)83.8% 83.8%   A  I  —             Y  S  G  S  T  Y  Y  S  P  S  L  K  S                            FR3 Name  66    67 68 69 70 71 72 73 74 75 76 77 78 79 80 Z14238|IGHV4-30-4*01   R     V  T  I  S  V  D  T  S  K  N  Q  F  S  L LpVH2-s2 (AM939769)   R     T  S  I  S  R  D  T  S  N  N  Q  F  S  L LpVH2-s3 (AM939770)   R     T  S  I  S  R  D  T  S  N  N  Q  F  S  L LpVH2-s4 (AM939771)   H     T  S  I  S  R  D  T  S  K  N  Q  F  S  L LpVH2-s5 (AM939772)   R     T  S  I  S  R  D  T  S  K  N  Q  F  S  L LpVH2-s6 (AM939773)   H     T  S  I  S  R  D  T  S  K  N  Q  F  S  L LpVH2-s11 Ps(AM939703)   R     T  S  I  S  R  D  T  S  K  N  Q  F  S  L LpVH2-s8 (AM939705)   R     T   S  I  S  R  D  T  S  N  N  Q  F  S  L LpVH2-s9 Ps(AM939706)   H     T  S  I  S  R  D  T  S  K  N  Q  F  S  L LpVH2-s10 (AM939702)   R     T  S  I  S  R  D  T  S  K  N  Q  F  S  L                              FR3Name  81    82 82a 82b 82c 83 84 85 86 87 88 89 90 91 92 Z14238|IGHV4-30-4*01   K     L      S  S   V   T  A  A  D  T  A  V  Y  Y  C LpVH2-s2 (AM939769)   Q     L  S  S   V   T  P  E  G  T  A  V  Y  Y  C LpVH2-s3 (AM939770)   Q     L  S  S   V   T  P  E  G  T  A  V  Y  Y  C LpVH2-s4 (AM939771)   Q     L  S  S   V   T  P  E  G  T  A  V  Y  Y  C LpVH2-s5 (AM939772)   Q     L  S  S   V   T  P  E  G  T  A  V  Y  Y  C LpVH2-s6 (AM939773)   Q     L  S  S   V   T  P  E  G  T  A  V  Y  Y  C LpVH2-s11 Ps(AM939703)  Q     L   S  S   V   T  P  E  G  T  A  V  Y  Y  C LpVH2-s8 (AM939705)  Q     L   S  S   V   T  P  E  G  T  C  V  Y  Y  CLpVH2-s9 Ps(AM939706)   Q     L  S  S   V   T  P  E  G  T  A  V  Y  Y  C LpVH2-s10 (AM939702)   Q     L  S  S   V   T  P  E  G  T  A  V  Y  Y  C Color codes Residue found inclosest matching germline Residue found in other germlines of the samesubclass (VH3, VK4, . . . ) Residue found in other germlines of the sameclass (VH, VK or VL) Residue found in any germline of the same class(VH, VK or VL) Primer-encoded residue Scores % Identity: fraction offramework residues which is found in the closest matching germline %Homology: fraction of framework residues which is found in the closestmatching germline or other germlines of the same subclass

10.11 Lama glama derived VH analysis (SEQ ID NOS: 326-332) Name % Ident% Homol 1 2 3 4 5 6 7 8 9 10 11 12 13 AJ879486|IGHV3-23*04 E V Q L V E SG G G L V Q S-VH1 88.5% 93.1% E V Q L V E S G G G L V Q S-VH3 87.4%90.8% E V Q L V Q S G G G L V Q S-VH4 92.0% 96.6% E E Q L V E S G G G LV Q L33851|IGHV3-74*01 E V Q L V E S G G G L V Q S-VH2 93.2% 95.5% E V QL V E S G G G L V Q S-VH6 93.1% 96.6% E V Q L V E S G G G L V Q S-VH593.1% 96.6% E V Q L V E S G G G L V Q Name % Ident % Homol 14 15 16 1718 19 20 21 22 23 24 25 26 AJ879486|IGHV3-23*04 P G G S L R L S C A A SG S-VH1 88.5% 93.1% P G G S L R L S C A A S G S-VH3 87.4% 90.8% H G G SL R L S C A A S G S-VH4 92.0% 96.6% P G G S L R L S C A A S GL33851|IGHV3-74*01 P G G S L R L S C A A S G S-VH2 93.2% 95.5% P G G S LR L S C A A S G S-VH6 93.1% 96.6% P G G S L R L S C A A S G S-VH5 93.1%96.6% P G G S L R L S C A A S G Name % Ident % Homol 27 28 29 30 31 3233 34 35 36 37 38 39 AJ879486|IGHV3-23*04 F T F S S Y A M S W V R QS-VH1 88.5% 93.1% F T F G R Y A M S W V R Q S-VH3 87.4% 90.8% F A F S SA G M S W V R Q S-VH4 92.0% 96.6% F T F G S Y D M Y W V R QL33851|IGHV3-74*01 F T F S S Y W M H W V R Q S-VH2 93.2% 95.5% F T F S SY Y M S W V R Q S-VH6 93.1% 96.6% F T F S S A V M S W V R Q S-VH5 93.1%96.6% F T F S S A V M S W V R Q Name % Ident % Homol 40 41 42 43 44 4546 47 48 49 50 51 52 AJ879486|IGHV3-23*04 A P G K G L E W V S A I SS-VH1 88.5% 93.1% A P G K G P E W V S A I S S-VH3 87.4% 90.8% A P G K GL E G V S A I N S-VH4 92.0% 96.6% A P G K G P E W V S A I RL33851|IGHV3-74*01 A P G K G L V W V S R I N S-VH2 93.2% 95.5% A P G K GL E W V S S I Y S-VH6 93.1% 96.6% A P G K G L E W V S G I G S-VH5 93.1%96.6% A P G K G L E W V S T I G Name % Ident % Homol 52a 53 54 55 56 5758 59 60 61 62 63 64 AJ879486|IGHV3-23*04 G S G G S T Y Y A D S V KS-VH1 88.5% 93.1% W N S G R I Y D A E S M K S-VH3 87.4% 90.8% T R S G TT Y Y A D F T K S-VH4 92.0% 96.6% S G G G S T Y Y A D S V KL33851|IGHV3-74*01 S D G S S T S Y A D S V K S-VH2 93.2% 95.5% S D G S YT Y Y A D S V K S-VH6 93.1% 96.6% S G G S T T S Y A D S V K S-VH5 93.1%96.6% A A G S T T S Y A D S V K Name % Ident % Homol 65 66 67 68 69 7071 72 73 74 75 76 77 AJ879486|IGHV3-23*04 G R F T I S R D N S K N TS-VH1 88.5% 93.1% G R F T V S R D N T K N T S-VH3 87.4% 90.8% G R F T IS R D N A K N T S-VH4 92.0% 96.6% G R F T I S R D N A K N TL33851|IGHV3-74*01 G R F T I S R D N A K N T S-VH2 93.2% 95.5% G R F T IS R D N A K N T S-VH6 93.1% 96.6% G R F T I S R D N A K N T S-VH5 93.1%96.6% G R F T I S R D N A K N T Name % Ident % Homol 78 79 80 81 82 82a82b 82c 83 84 85 86 87 AJ879486|IGHV3-23*04 L Y L Q M N S L R A E D TS-VH1 88.5% 93.1% L Y L Q M N A L K T D D T S-VH3 87.4% 90.8% V Y L Q MN S L K P E D T S-VH4 92.0% 96.6% L Y L Q M N S L K P E D TL33851|IGHV3-74*01 L Y L Q M N S L R A E D T S-VH2 93.2% 95.5% L Y L Q MN S L K S E D T S-VH6 93.1% 96.6% L Y L Q M N S L K P E D T S-VH5 93.1%96.6% L Y L Q M N S L K P E D T Name % Ident % Homol 88 89 90 91 92 9394 95 96 97 98 99 100 AJ879486|IGHV3-23*04 A V Y Y C A K S-VH1 88.5%93.1% A V Y Y C A R S T A E S N S-VH3 87.4% 90.8% A V Y Y C N A G F P SS-VH4 92.0% 96.6% A V Y Y C A K P S T I A T L33851|IGHV3-74*01 A V Y Y CA R S-VH2 93.2% 95.5% A V Y Y C A N W D Y S G S S-VH6 93.1% 96.6% A V YY C T G R G F S-VH5 93.1% 96.6% A V Y Y C T G R G F Name % Ident % Homol100a 100b 100c 100d 100e 100f 100g 101 102 103 104 105 106AJ879486|IGHV3-23*04 S-VH1 88.5% 93.1% W I P L D W G Q G S-VH3 87.4%90.8% R Y W G Q G S-VH4 92.0% 96.6% I L F T S W G Q G L33851|IGHV3-74*01S-VH2 93.2% 95.5% Y Y A P A T F G S W G Q G S-VH6 93.1% 96.6% S S W G QG S-VH5 93.1% 96.6% S S W G Q G Name % Ident % Homol 107 108 109 110 111112 113 AJ879486|IGHV3-23*04 S-VH1 88.5% 93.1% T Q V T V S S S-VH3 87.4%90.8% T Q V T V S S S-VH4 92.0% 96.6% T Q V T V S S L33851|IGHV3-74*01S-VH2 93.2% 95.5% T R V T V S S S-VH6 93.1% 96.6% T Q V T V S S S-VH593.1% 96.6% T Q V T V S S Chart codes Residue found in closest matchinggermline Residue found in other germlines of the same subclass (VH3,VK4, . . . ) Residue found in other germlines of the same class (VH, VKor VL) Residue not found in any germline of the same class (VH, VK orVL) Primer-encoded residue Scores % Identity: fraction of frameworkresidues which is found in the closest matching germline % Homology:fraction of framework residues which is found in the closest matchinggermline or other germlines of the same subclass

10.12 Lama glama derived VL analysis (SEQ ID NOS: 333-346) Lambda                                                    2 2 2  %                  1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 7 7 7 2 2 3 3 3 3 3Name Homol1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 a b c 8 9 0 1 2 3 4D86994|IGLV3-25*02 S Y E L T Q P P S V S V S P G Q T A R I T C 

          

VL-25-28 91.3%N F M L T Q P S A L S V T L G Q T A K I T C Q G G S       L G S S Y A H                                  5 5 5 5 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 5 5 1 1 1 1 5 5 5 5 5 5 5 5 6 6  Name %5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 a b c d 2 3 4 5 6 7 8 9 0 1D86994|IGLV3-25*02 Homol W Y Q Q K P G Q A P V L V I Y 

          

     G I P E R VL-25-28 91.3%W Y Q Q K P G Q A P V L V I Y D D         D S R P S G I P E R                                                          2 2 2 %                  1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 7 7 7 2 2 3 3 3 3 3Name Homol1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 a b c 8 9 0 1 2 3 4Z73672|IGLVS-37*01 Q P V L T Q P P S S S A S P G E S A R L T C 

VL2, 12, 15 97.5%Q A V L T Q P P S L S A S P G S S V R L T C T L S S G N S V G S Y D I S                                   5 5 5 5  %3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 5 5 1 1 1 1 5 5 5 5 5 5 5 5 6 6  NameHomol 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 a b c d 2 3 4 5 6 7 8 9 0 1Z73672|IGLVS-37*01 W Y Q Q K P G S P P R Y L L Y 

           G V P S R  VL2, 12, 15 97.5%W Y Q Q K A G S P P R Y L L Y Y Y S D S F N H Q G S G V P S R                                                    2 2 2 %                  1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 7 7 7 2 2 3 3 3 3 3Name Homol1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 a b c 8 9 0 1 2 3 4Z73650|IGLV8-61*01 Q T V V T Q E P S F S V S P G G T V T L T C 

VL3, 5 90.0%Q A V V T Q E P S L S V S P G G T V T L T C G L S S G S V T S S N Y P GVL17-24, 29-32 90.0%Q A V V T Q E P S L S V S P G G T V T L T C G L S S G S V T T S N Y A A VL10 91.3%S S E L T Q E P S L S V S P G G T V T L T C G L S S G S V T S S N Y P GVL4, 6, 7, 8, 9, 97.5%S Y E L T Q D P S L S V S P G E T V T L T C G L N S G S V T S H N Y P A13, 14                                   5 5 5 5 %3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 5 5 1 1 1 1 5 5 5 5 5 5 5 5 6 6 Name Homol5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 a b c d 2 3 4 5 6 7 8 9 0 1Z73650|IGLV8-61*01 W Y Q Q T P G Q A P R T L I Y 

         

      G V P D R   VL3, 5 90.0%W Y Q Q K P G Q A P R T L I Y N T         N S R Y S G V P N RVL17-24, 29-32 90.0%W F Q Q A P G Q A P R T L I Y K T         N S R H S G V P S R VL10 91.3%W Y Q Q K P G Q A P R T L I Y N T         N S R Y S G V P N R VL4, 6, 7, 8, 9, 97.5%W Y Q Q K P G Q A P R T L M Y N T         N S R Y P M V P P R 13, 14                6 6 %6 6 6 6 6 6 6 6 8 8 6 7 7 7 7 7 7 7 7 7 7 8 8 8 8 8 8 8 8 8  Name Homol1 2 3 4 5 6 7 8 a b 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8D86994|IGLV3-25*02R F S G S S S G     T T V T L T I S G V Q A E D E A D Y Y C  VL25-2891.3% R F S G S S S G     G R A T L T I S G A Q A E D E G D Y Y C              9 9 9         1 1 1 1 1 1 1 1 %8 9 9 9 9 9 9 5 5 5 9 9 9 9 0 0 0 0 0 0 0 0 Name Homol9 0 1 2 3 4 5 a b c 6 7 8 9 0 1 2 3 4 5 6 7 D86994|IGLV3-25*02

             

VL25-28 91.3% Q S A D S S G N     A V F G G G T H L T V L                 6 6 %6 6 6 6 6 6 6 6 8 8 6 7 7 7 7 7 7 7 7 7 7 8 8 8 8 8 8 8 8 8 Name Homol1 2 3 4 5 6 7 8 a b 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8Z73672|IGLV5-37*01R F S G S K D A S A N T G I L L I S G L Q S E D E A D Y Y C VL2, 12, 1597.5% R F S G S K D A S A N A G L L L I S G L Q P E D E A D Y Y C              9 9 9         1 1 1 1 1 1 1 1 %8 9 9 9 9 9 9 5 5 5 9 9 9 9 0 0 0 0 0 0 0 0 Name Homol9 0 1 2 3 4 5 a b c 6 7 8 9 0 1 2 3 4 5 6 7 Z73672|IGLV5-37*01

             

VL2, 12, 15 97.5% S A Y K S G S Y N   P T F G G G T K L T V L                6 6 %6 6 6 6 6 6 6 6 8 8 6 7 7 7 7 7 7 7 7 7 7 8 8 8 8 8 8 8 8 8 Name Homol1 2 3 4 5 6 7 8 a b 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8Z73650|IGLV8-61*01R F S G S I L G     N K A A L T I T G A Q A D D E S D Y Y C VL3, 5 90.0%R F S G S I S G     N K A V L T I T G A Q P E D E A D Y Y C VL17-24, 29-32 90.0%R F S G S I S G     N K A A L T I T G A Q P E D E A D Y Y C VL10 91.3%R F S G S I S G     N K A A L T I T G A Q P E D E A D Y Y CVL4, 6, 7, 8, 9,  97.5%R F S G S I S G     M K A A L T I T G A Q A E D E A D Y Y C 13, 14              9 9 9         1 1 1 1 1 1 1 1 %8 9 9 9 9 9 9 5 5 5 9 9 9 9 0 0 0 0 0 0 0 0 Name Homol9 0 1 2 3 4 5 a b c 6 7 8 9 0 1 2 3 4 5 6 7 Z73650|IGLV8-61*01

             

  VL3, 5 90.0% A V Y T G S S N Y P A V F G G G T H L T V LVL17-24, 29-32 90.0% S L Y P G S D I     S V F G G G T H L T V L VL1091.3% A V Y I G S G G Y P P V F G G G T K L T V L VL4, 6, 7, 8, 9, 97.5%A V Y I R S R T     L E F G G G T H L T V L 13, 14 Kappa % %                  1 1 1 1 1 1 1 1 1 1 2 2 2 2 Name Ident Homol1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3  U41644|IGHV2D-29*02D I V M T Q T P L S L S V T P G Q P A S I S C KAPPA 33-36, 38, 39 86.3%90.0% D I V M T Q T P G S L S V V P G E S A S I S C   42, 4KAPPA 41, 43 44 86.3% 90.0%D I V M T Q T P G S L S V V P G E S A S I S C KAPPA 40, 44 86.3% 90.0%D I V M T Q T P G S L S V V P G E S A S I S C KAPPA 37, 46, 48 83.3%90.0% E I V L T   T P G S L S V V P G E S A S I S C         2 2 2 2 2 %% 2 2 2 2 7 7 7 7 7 2 2 3 3 3 3 3 Name Ident Homol4 5 6 7 a b c d e 8 9 0 1 2 3 4  U41644|IGHV2D-29*02

KAPPA 33-36, 38, 39 86.3% 90.0% K A S Q S L V H S D G K T Y L Y 42, 4K A S Q S L V H S D G K T Y L Y KAPPA 41, 43 44 86.3% 90.0%K A S Q S L V L S G G K T Y L Y KAPPA 40, 44 83.3% 90.0%K A S Q S L V R S D G K T Y L Y KAPPA 37, 46, 48 86.3% 90.0% % %3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 5 5 5 Name Ident Homol5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 U41644|IGKV2D-29*02W Y L Q K P G Q S P Q L L I Y 

       G V P KAPPA 33-36, 38, 86.3% 90.0%W L L Q K P G Q S P Q R L I Y Q V S N R G S G V P   39, 42, 4KAPPA 41, 43, 44 86.3% 90.0%W L L Q K P G Q S P Q R L I Y Q V S N R G S G V P KAPPA 40, 44 86.3%90.0% W L L Q K P G Q S P Q R L I Y Q V S N R G S G V P KAPPA 37, 46, 4886.3% 90.0% W L L Q K P G Q S P Q R L I Y Q V S N R G S G V P

Example 11 Analysis of Key Residues for Canonical Folds of H1 and H2 andComparison of H1 and H2 Residues with Human Germline

Structural analysis of antibodies revealed the relationship between thesequence and the shape of the binding site formed by the complementaritydetermining regions (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987);Tramontano et al., J. Mol. Biol. 215:175-82 (1990)). Despite their highsequence variability, five of the six loops adopt just a smallrepertoire of main-chain conformations, called “canonical structures”.These conformations are first of all determined by the length of theloops and secondly by the presence of key residues at certain positionsin the loops and in the framework regions that determine theconformation through their packing, hydrogen bonding or the ability toassume unusual main-chain conformations.

We have analyzed the predicted canonical structures of H1 and H2 for thegermline dromedary and llama VH segments based on the length of theseloops and the presence of the previously mentioned key residues. Thecomparison was made with the key residues as they occur in the closestmatching human germline in terms of the presence of identicalcombination of canonical folds and overall sequence homology (Table 1);in addition the amino acids compatible with the corresponding canonicalfold as proposed by Morea and colleagues (Morea et al., Methods20:267-279 (2000)) are shown. For the dromedary germline VH familyIGHV1S(1-19), which has canonical fold 1 for H1 (coded as H1: 1 inTable 1) and fold 1 for H2 (H2: 1), and family IGHV1 S(20, 22, 23, 24)with fold 1 for H1 (H1: 1) and fold 3 for H2 (H2: 3), and familyIGHV1S(21, 25-39) with fold 1 for H1 (H1: 1) and fold 2 for H2 (H2: 2)and llama germline IGHV1S8 with fold 1 for H1 (H1: 1) and fold 2 for H2(H2: 2), the key residues 24, 26, 27, 29, 34 and 94 for the canonicalfold of H1 are shown along with these from the analogue human germlinefamily with the same canonical fold combination (upper part of Table 1).Also for dromedary germline VH family IGHV1S(1-19) with fold 1 for H1(H1: 1) and fold 1 for H2 (H2: 1), and family IGHV1 S(20, 22, 23, 24)with fold 1 for H1 (H1: 1) and fold 3 for H2 (H2: 3), and family IGHV1S(21, 25-39) with fold 1 for H1 (H1: 1) and fold 2 for H2 (H2: 2) andllama germline IGHV1 S8 with fold 1 for H1 (H1: 1) and fold 2 for H2(H2: 1) the key residues 52a or 54 or 55 in combination with residue 71are shown along with these from the analogue human germline familieswith the same canonical fold combinations (lower part Table 1).

The analysis clearly demonstrates the “human nature” of the canonicalloops, since the key residues found in the camelid VH segments areidentical to what is found in the corresponding human VH segments. Forexample, all 19 germline VH segments from dromedary coded IGHV1S(1-19)have Alanine on position 19, Glycine on 26, Phenylalanine on 27 and 29and Methionine on 34 as they predominantly occur in human germlinefamily 3 members, which like these dromedary germline sequences have acanonical fold type 1 for H1 and fold type 1 for H2. Position 94 isencoded only in a single germline dromedary (out of the total of 39germline VH segments) meaning that a proper analysis is not possible.Nguyen and colleagues (Nguyen et al., EMBO J. 19:921-930 (2000)) noticedthat the dromedary germline VH and VHH segments “span from the conservedoctamer (i.e. recombination signal) to the Cysteine residue 92 of FR3”(end of citation), whereas the human segments encode two additionalresidues (93 and 94). However, we will discuss this residue during theanalysis of the only six known Somatically mutated VH segments derivedfrom llama derived conventional antibodies.

There are virtually no exceptions for the perfect match with the humangermline segments and the key residues proposed by Morea and colleagues(Morea et al., Methods 20:267-279 (2000)), besides residue 52a for llamagermline IGHV1 S6 for canonical fold type 2 for loop H2, which hasSerine on this position, while the human analogue uses Threonine. It isencouraging to observe that in four of the six published llama VHderived from somatically mutated conventional antibodies (Vu et al.,Mol. Immunol. 34:1121-31 (1997) Threonine is found on position 52a. Itis worthwhile to mention that Serine and Threonine are closely related,since they both have a polar hydroxyl group and small sidegroups,suggesting that it might be possible to exchange both residues duringhumanization.

Glutamine on position 71, which occurs in 1 out of 19 dromedary germlineVH segments with a canonical fold type 1 for loop H2 and in 4 out of 16dromedary germline VH with a canonical fold type 2 for H2, seems to berather extraordinary. The H2 loop packs against the residue at site 71and the position of the loop relative to the framework is mainlydetermined by the size of the residue at this site. Canonical structures2 and 3 are found in H2 loops with 6 residues. Structure 2 occurs whenresidues 52a and 71 are small or medium sized hydrophobic residues,while canonical fold 3 occurs when residue 71 is Arginine or Lysine. Itcan not be predicted how often the dromedary germline segments withGlutamine on position 71 will be used in somatically mutatedconventional antibodies, but the humanization of this residue inantibody leads with this particular residue needs to be carefullyexamined. The presence of Arginine and Glutamine on position 71 in thedromedary IGHV1 S(20, 22, 23, 24) family members with canonical fold 2for H2 is rather unexpected, but on the other hand human germline VH1family members VH1-9, VH1-10 and VH1-11 with canonical fold 2 for H2have Arginine as well, while VH5 member 5-1 with fold 2 for H2 carriesGlutamine on this position.

As Chothia and colleagues did when discussing the structural repertoireof the human VH segments (Chothia et al., J. Mol. Biol. 227:799-817(1992)) we examined the individual amino acid residues of the H1 and H2loops of the dromedary and llama VH segments along with the key residuesand compared these with the human counterparts having the same canonicalfold combination (Table 2). For dromedary germline VH familyIGHV1S(1-19) with canonical fold 1 for H1 and fold 1 for H2, and familyIGHV1 S(20, 22, 23, 24) with fold 1 for H1 (H1: 1) and fold 3 for H2(H2: 3), and family IGHV1S(21, 25-39) with fold 1 for H1 (H1: 1) andfold 2 for H2 (H2: 2) and llama germline IGHV1S8 with fold 1 for H1 andfold 2 for H2 the H1 residues 26 to 33 and the are shown together withthe key residues 24 and 94 located outside of H1 (Table 2A). Inaddition, for dromedary germline VH family IGHV1S(1-19) with canonicalfold 1 for H1(H1: 1) and fold 1 for H2 (H2: 1), and family IGHV1 S(20,22, 23, 24) with fold 1 for H1 (H1: 1) and fold 3 for H2 (H2: 3), andfamily IGHV1 S(21, 25-39) with fold 1 for H1 (H1: 1) and fold 2 for H2(H2: 2) and llama germline IGHV1 S8 with fold 1 for H1 (H1: 1) and fold2 for H2 (H2: 2) the H2 residues 52 to 56 were analyzed with key residue71, which is located outside H2 (Table 2B).

It is surprising to see the very high degree of sequence homology in thevariable loops, especially in H1 there are hardly differences with therelevant human sequences. For instance, germline dromedary familyIGHV1S(1-19) with canonical fold combination H1: 1/H2: 1 containspredominantly Alanine on position 24, Glycine on 26, Phenylalanine on27, Threonine on 28, Phenylalanine on 29, Serine on 30 and 31, Tyrosineon 33, Methionine on 34 and Serine on 35, which completely match thehuman germline family 3 members that share the same combination of H1and H2 canonical folds. Exceptions are residue 27 (Phenylalanine) and 32(Serine) of the only publicly known llama germline VH segment, but againin 4 out of 6 somatically mutated llama VH which are publicly known (Vuet al., Mol. Immunol. 34:1121-1131 (1997)). Tyrosine is present on 32 asis found in the analogue human germlines. The same high degree ofsequence homology is found for the H2 loops of dromedary germline VHsegments with the exception of residue 54 of family IGHV1S(1-19).Especially dromedary family IGHV1S(21, 25-39) deviates on a numberpositions in the H2 loop (i.e. 53, 54, 56 and 58). The H2 loop of thellama germline VH segment with the same fold scores much better, but aswell contains a number of deviating residues on position 50, 52, 52a,54, 55 and 58, but the interpretation is rather difficult, since theanalysis was performed with the only known germline segment. Theanalysis of somatically mutated VH derived from llama shows that certainresidues on these positions occur, which also appear in thecorresponding human germline sequences, although infrequently (f.i.Glycine on 50, Asparagine on 52 and 58, Threonine on 52a and Glycine on55).

We also analyzed the panel of six publicly known somatically mutated VHsequences from llama (Vu et al., Mol. Immunol. 34:1121-31 (1997)). Belowthe alignment with human VH3 member 3-23 is shown, demonstrating a veryhigh degree of sequence homology: overall, only 3 deviating residueswere observed, one of which is encoded by the primer used foramplification, while the other two occur in human germlines of the sameclass. Even the CDRs show to have a very high degree of sequencehomology: CDR1 is probably identical, while only three residues of CDR2are different. Canonical fold analysis reveals that two VH have fold 1for H1 and fold 2 for H2, as was observed for the only available llamaderived germline VH, but the other four have fold 1 for H1 and fold 3for H2 as occurs in 3-23 and the majority of the human family VH3germline segments. This might be suggesting that these are derived fromother, not yet known germline VH segments. Examination of the keyresidues supporting the canonical folds gives the perfect match withthose occurring in human germline with the same canonical foldcombination as was already observed for the llama germline VH segmentlisted in Table 1. It is very interesting to see that key residue 94 inthese somatically mutated sequences is Lysine (2 out of 5), Serine (1out of 5) and Arginine (1 out of 5), which are all found in the humangermlines with the same fold combination or are proposed by Morea andcolleagues (Morea et al., Methods 20:267-279 (2000)).

11.1 Somatically mutated Llama VH from conventional antibodies (SEQ ID NOS: 104-110)IGHV          FR1-IMGT         CDR1-IMGT        FR2-IMGT       CDR2-IMGTgene           (1-26)           (27-38)         (39-55)         (56-65)1       10        20         30         40        50         60 .........|.........|...... ...|........ .|.........|..... ....|.....M99660, IGHV3-23EVQLLESGG.GLVQPGGSLRLSCAAS GFTFSSYA.... MSWVRQAPGKGLEWVSA ISGSGGST..IVH28EVQLVESGG.GLVQPGGSLRLSCAAS GFAFSSYD.... MSWVRQAPGKGLEWVSA INSGGIST..IVH69EVQLVESGG.GLVQPGRSLRVSYAAS GFTFSSHY.... MSWVRQDPEKGLEWVSE IATGGTIT..IVH47EVQLVESGG.GLVQPGGSLRLSCAAS GLTFDDYA.... MSWVRQAPGKGLEWVST IYTHSRNT..IVH48EVQLVESGG.GLVQPGGSLRLSCAAS GFTFSSYV.... MSXVRQAPGKGPEWVSG VNTDGRSI..IVH70EVQLVESGG.GLVQPGGSLRVSCAAS GFTFSSLY.... MSWVRQVPGKGLEWVST IHTASGST..IVH71EVQLVESGG.GLVQPGGSLRLSCAAS GFTFSSYD.... MSWVRQAPGKGLEWVSG IYSDGTTT..IGHV                 FR3-IMGT               CDR3-IMGT gene                (66-104)               (105-115)   70        80        90        100       110 ....|.........|.........|.........|.... .....|.....   (J1, 4, 5)M99660, IGHV3-23YYADSVK.GRFTISRDNSKNTLYLQMNSLRAEDTAVYYC AK.........     WGQGTLVTVSSIVH28YNADSMK.GRFTISRDNAKNTVYLQMNSLKPEDTAVYYC NADTWYCDQLDSSDY WGQGTQVTVSSIVH69SYADSVK.GRFTISRDNANNMLFLQMNNLKPEDTALYYC VRRGRAIA....FDV WGQGTLVTVSSIVH47YYADSVK.GRFTISRDNAKNTLYLQMNSLKSDDTALYYC AKEWVGSVVEGRYRG WGQGTLVTVSSIVH48TYADSVK.GRFTISRDNAKNTLYLQMNSLKPEDTAVYYC TKICTVITGRPGYDY WGQGTLVTVSSIVH70FYADSVQ.GRFLVSRDNAKNTLYLQMDSLKPEDTARYYC ASAILGQ.....YDY WGQGTLVTVSSIVH71YDGDSVK.GRFTISRDNAKNMLYLQMNSLKPEDAAVYYC ASAIRGW.....YDY WGQGTLVTNSSA) Sequence homology:1) position 5 Valine (primer encoded) also found in f.i. IGHV3-152) position 83 Alanine is found more often in human VH3 germlines then Serine of 3-233) position 95/96 Lysine/Proline occurs in IGHV3-15/49/73 as Lysine/Threonine4) CDR1 is completely human-like, in CDR2 only residues 57, 58 and 59 seem to deviatefrom human germline 3-23 (57 Serine, 58 Glycine, 59 Serine)5) FR4 gives perfect match with human J1, J4 and J5B) Canonical fold analysis1) combination fold 1 for H1 and fold 2 for H2 for IVH28 and 1VH69 (as in 3 out of11 human germline family VH1 members and both human germline family VH5 members)2) for IVH47, 48 70 and 71 combination fold 1 for H1 and fold 3 for H2 as found in3-23 and majority of family VH3 members Examples of analysis: IVH28:CDR H1 Class ? ! Similar to class 1/10A, but: !H33 (Chothia Numbering) = D (allows: YAWGTLV) !H94 (Chothia Numbering) = A (allows: RKGSHN) CDR H2 Class ? !Similar to class 2/10A, but: ! H33 (Chothia Numbering) =D (allows: YWGATL) ! H59 (Chothia Numbering) = N (allows: Y) !H71 (Chothia Numbering) = R (allows: VAL) IVH47:CDR H1 Class 1/10A [2fbj] CDR H2 Class ? ! Similar to class 3/10B, but:! H52 (Chothia Numbering) = Y (allows: SFWH)  !H53 (Chothia Numbering) = H (allows: DGSN)

Example 12 Analysis Key Residues for Canonical Folds of L1(λ) and L2(λ)and Comparison of L1(λ) and L2(λ) Residues with Human Germline

Also the predicted canonical structures of L1 and L2 were analyzed forthe somatically mutated dromedary VLambda segments based on length ofthe loops and the presence of the key residues relevant for thecanonical folds. The comparison was made with the key residues occurringin the closest matching human germline with the same combination ofcanonical folds and overall sequence homology (Table 3). For thesomatically mutated dromedary VL family VL3-1 (Camvl8, 18, 19, 20 and23), with fold 11 for L1 (L1: 11) and fold 7 for L2 (L2: 7), andsomatically mutated dromedary VL family VL3-12/32 (Camvl11) with fold 11for L1 (L1: 11) and fold 7 for L2 (L2: 7), and somatically mutateddromedary VL1-40 (Camvl44) with fold 14 for L1 (L1: 14) and fold 7 forL2 (L2: 7), and somatically mutated dromedary VL2-18 (Camvl5, 17, 30-33,36, 52, 57, 59, 60 and 65) with fold 14 for L1 (L1: 14) and fold 7 forL2 (L2: 7) the key residues 2, 25, 29, 30, 33 and 71 relevant for thecanonical fold of L1 are shown along with these from the analogue humangermline family with the same canonical fold combination (upper part ofTable 3). For somatically mutated dromedary VL family VL3-1 (Camvl8, 18,19, 20 and 23), with fold 11 for L1 (L1: 11) and fold 7 for L2 (L2: 7),and somatically mutated dromedary VL family VL3-12/32 (Camvl11) withfold 11 for L1 (L1: 11) and fold 7 for L2 (L2: 7), and somaticallymutated dromedary VL1-40 (Camvl44) with fold 14 for L1 (L1: 14) and fold7 for L2 (L2: 7), and somatically mutated dromedary VL2-18 (Camvl5, 17,30-33, 36, 52, 57, 59, 60 and 65) with fold 14 for L1 (L1: 14) and fold7 for L2 (L2: 7) the key residues 48 and 64 important for the canonicalfold of L2 are shown along with the key residues from the analogue humangermline families having the same canonical fold combinations (lowerpart Table 3).

As observed for VH the analysis reveals the “human nature” of bothcanonical loops L1 and L2, because here also the key residues of thecamelid VLambda segments are identical to those of the correspondinghuman VLambda segments. For example, in dromedary VL3-12/32, VL1-40 andVL2-18 all key residues of L1 are identical to those occurring in thecorresponding human germlines, and in dromedary VL3-1, VL3-12/32 andVL2-18 the key residues of L2 completely match with the correspondinghuman germline VL segments. Basically there are only two exceptions.First of all, L2 key residue 64 of VL1-40 is Glutamate, which is ratherdifferent from Glycine that is present in human germline Vλ1 with thesame L1/L2 combination of canonical folds as VL1-40. It is difficult todraw general conclusions, since VL1-40 only consists of an orphan (i.e.Camvl44). The second exception is VL3-1, where Phenylalanine is the mostdominantly occurring L1 key residue on position 30, whereas Leucine isfrequently found in human Vλ family 3 members that share fold 11 for L1and fold 7 for L2 with VL3-1. However, Leucine is also present in oneout of the five VL3-1 members.

We analyzed the individual amino acid residues of the L1 and L2 loops ofthe somatically mutated dromedary VLambda segments along with the keyresidues and made the comparison with the human counterparts sharing thesame canonical fold combination (Table 4). For the somatically mutateddromedary VL family VL3-1 (Camvl8, 18, 19, 20 and 23), with fold 11 forL1 (L1: 11) and fold 7 for L2 (L2: 7), and somatically mutated dromedaryVL family VL3-12/32 (Camvl11) with fold 11 for L1 (L1: 11) and fold 7for L2 (L2: 7), the L1 residues 27 to 33 together with key residue 71outside L1 are compared with the same residues present in thecorresponding human germline Vλ with same combination of folds for L1and L2 (upper part of Table 4A). For dromedary somatically mutateddromedary VL1-40 (Camvl44) with fold 14 for L1 (L1: 14) and fold 7 forL2 (L2: 7), and somatically mutated dromedary VL2-18 (Camvl5, 17, 30-33,36, 52, 57, 59, 60 and 65) with fold 14 for L1 (L1: 14) and fold 7 forL2 (L2: 7) the L1 residues 26 to 33 together with key residues 2 and 71outside L1 are compared with the same residues present in thecorresponding human germline Vλ family with same combination of foldsfor L1 and L2 (lower part of Table A). In addition, for somaticallymutated dromedary VL family VL3-1 (Camvl8, 18, 19, 20 and 23), with fold11 for L1 (L1: 11) and fold 7 for L2 (L2: 7), and somatically mutateddromedary VL family VL3-12/32 (Camvl11) with fold 11 for L1 (L1: 11) andfold 7 for L2 (L2: 7), and somatically mutated dromedary VL1-40(Camvl44) with fold 14 for L1 (L1: 14) and fold 7 for L2 (L2: 7), andsomatically mutated dromedary VL2-18 (Camvl5, 17, 30-33, 36, 52, 57, 59,60 and 65) with fold 14 for L1 (L1: 14) and fold 7 for L2 (L2: 7) the L2residues 49 to 53 together with key residues 48 and 64 located outsideL2 are compared with the same residues present in the correspondinghuman germline Vλ family with the same combination of canonical foldsfor L1 and L2 (Table 4B). There is a high degree of sequence homologybetween the L1, L2 and key residues with the human germline sequencesand only few exceptions exist, which mainly can be found in the orphanmembers of the VL3-12/32 and VL1-40 families. For L1 of VL3-12/32residues 27, 28, 30, 30a and 30b deviate from the corresponding humangermline Vλ family 3, while for the same loop residues 30b, 31 and 32 ofVL1-40 are different from the matching human germline Vλ family 1. Inthe orphan member of VL3-12/32 L2 residue 50 differs from the humananalogue, while for the only member of VL1-40 key residue 64 differsfrom the human analogue. A fundamental difference can be observed for L1residue 28 of dromedary family VL2-18 (Asparagine or Glutamate versusSerine in analogue human Vλ family 2).

Example 13 Analysis Key Residues for Canonical Folds of L1(κ) and L2(κ))and Comparison of L1(κ)) and L2(κ)) Residues with Human Germline

The predicted canonical structures of L1(κ)) and L2(κ)) were analyzedfor the somatically mutated dromedary VKappa segments based on looplength and presence of key residues. As before the comparison was madewith the key residues occurring in the closest matching human germlinewith the same combination of canonical folds and overall sequencehomology (Table 5). For the somatically mutated dromedary VK familyVK2-40 (Kp1, 3, 6, 7, 10, 20 and 48), with fold 3 for L1 (L1: 3) andfold 1 for L2 (L2: 1) the key residues 2, 25, 29, 30e, 33 and 71relevant for the canonical fold of L1 are shown along with these fromthe analogue human VKappa germline family 2 with the same canonical foldcombination. In addition the key residues compatible with thecorresponding canonical fold as proposed by Morea et al. are shown inthe bottom line. There is a perfect match for key residues 2, 25, 33 and71 and to a certain degree for residue 29. Residue 30e of the dromedaryVKappa is Glutamine in stead of Glycine, but Morea and colleagues (Moreaet al., Methods 20:267-279 (2000)) suggested this residue to beperfectly compatible with the fold 3 for L1.

For the same somatically mutated dromedary VK family VK2-40 (Kp1, 3, 6,7, 10, 20 and 48) with fold 3 for L1 (L1: 3) and fold 1 for L2 (L2: 1)the key residues 48 and 64 determining the canonical fold of L1 areshown together with these from the analogue human VKappa germline family2. Here again the match is perfect.

The individual amino acid residues of the L1 and L2 loops of thesomatically mutated dromedary VKappa segments along with the keyresidues were compared with those occurring in the human counterpart (VKfamily 2) that shares the same canonical fold combination (Table 6). Forthe somatically mutated dromedary VK family VK2-40 (Kp1, 3, 6, 7, 10, 20and 48) with fold 3 for L1 (L1: 3) and fold 1 for L2 (L2: 1) the L1 andL2 residues were compared with those found in germline VK family 2 thathas the identical canonical loop combination. The majority of theresidues are shared between the dromedary somatically mutated VK and thehuman germline, such as Isoleucine on position 2, Serine on 25, 26 28and 30b, Glutamine on 27, Tyrosine on 32, Leucine on 33 andPhenylalanine on 71. However, a few differ from the human analogue, i.e.Valine on 29 (although Leucine of human germline also occurs in thedromedary VK), Phenylalanine on 30 (again human residue Leucine is foundas well in dromedary VK), Serine on 30a (human residue Glutamate occursinfrequently in dromedary VK), Serine on 30c, Asparagine on 30d,Glutamine on 30e, Lysine on 30f and finally Serine on 31.

Together with the key residues the residues of the L2 loop of thesomatically mutated dromedary VKappa segments were compared thoseoccurring in the human counterpart (VK family 2) that shares the samecanonical fold combination (Table 6). Here again a perfect match wasobserved from residue Isoleucine on 48, Tyrosine on 49, Serine on 52 andGlycine on 64, while deviations were found on position 50 (Tyrosine instead of Threonine from human VK family 2), and 51 (Alanine on 51instead of Leucine).

Overall Conclusion

This analysis of the camelid VH and VL sequences demonstrates a veryhigh homology if not identity to the human key residues defining thecanonical folds as well as to the residues found in the hypervariableloops themselves. This suggests that the vast majority of the camelidimmunoglobulin sequences will adopt the canonical folds as found in thehuman germlines, not only for the individual hypervariable loops but aswell as for the combination of canonical folds found in human VH and VL.

TABLE 1 Sites of key residues for determining canonical folds ingermline dromedary and llama VH. Canonical Closest hu H1 key residues H2key residues Sequence structure GL family 24 26 27 29 34 94 52a 54 55 71GI Drom IGHV1S H1: 1 (3-53) A(19) G(19) F(19) F(19) M(19) G(1); -(18)(1-19) Hu GI family 3 A(23); G(24) F(24) F(22); M(23); R(18); G(1) V(2)T(1) K(5); T(1) GI Drom IGHV1S H1: 1 (3-23) A(4) G(4) F(4) F(4) M(4) —(20, 22, 23, 24) Hu GI family 3 A(23); G(24) F(24) F(22); M(23); R(18);G(1 ) V(2) T(1) K(5); T(1) GI Drom IGHV1S H1: 1 (V_(I-4, 1b)) A(16) G15;F(16) F(14); M(13); — (21, 25-39) A(1) S(1); I(2); Y(1) V(1) Hu GIfamily 1 A(19); G(21) Y(18); F(20); M(13); R(17); V(2) F(1); L(1) I(5);T(2); G(2) L(2); A(1) V(1) GI Llama IGHV1S6 H1: 1 (3-23) A(1) G(1) F(1)F(1) M(1) A(1) Hu GI family 1 A(19); G(21) Y(18); F(20); M(13); R(17);V(2) F(1); L(1) I(5); T(2); G(2) L(2); A(1) V(1) H1: 1 A; V; G F; Y; F;L; M; V; R; G; (Morea) S; T S; D I I; Y; W N; K; S GI Drom IGHV1S H2: 1(3-53) — — G(19) R(18); (1-19) Q(1) Hu GI family 3 — — G(4) R(4) H2. 1G, D K, R, (Morea) V, I GI Drom IGHV1S H2: 3 (3-23) — G(4) — R(4) (20,22, 23, 24) Hu GI family 3 — G(13); — R(14) S(3) H2: 3 — G; S; — R; K(Morea) N; D GI Drom IGHV1S H2: 2 (V_(I-4, 1b)) S(10); — G(16) R(8);(21, 25-39) T(6) K(4); Q(4) Hu GI family 1 P(3); — G(7) A(3); T(2);T(2); A(2) L(2) H2: 2 P; T; G; N; A; L; (Morea) A D; S T GI LlamaIGHV1S6 H2: 2 (3-23) S(1) — S(1) T(1) Hu GI family 1 P(3); — G(7) A(3);T(2); T(2); A(2) L(2) H2: 2 P; T; G; N; A; L; (Morea) A D; S T Numberbetween brackets indicates frequency of residue as found indromedary/llama or human germline; numbering of key residues accordingto Kabat et al. (Sequences of Proteins of Immunological Interest, 5^(th)ed. (1991)) and key residues proposed by Morea and colleagues. (Morea etal., Methods 2000). Ha: b indicates canonical fold type b for loop Ha.

TABLE 2(A) Comparison of H1 sequences for dromedary and llama germlineVH with human germlines; numbering according to Kabat et al. (Sequencesof Proteins of Immunological Interest, 5th ed. (1991)). H1: 1 residueno. 2 2 2 2 2 3 3 3 3 3 3 3 3 9 4 6 7 8 9 0 1 1a 1b 2 3 4 54 * * * * * * GI Drom IVHG1S A(19) G(19) F(19) T(19) F(19) S(19) S(19) —— Y(19) Y(14); M(19) S(17); G(1) (1-19) D(2); Y(2) W(2); A(1) Family 3Hu GL A(3); G(4) F(4) T(4) V(2); S(4) S(4) — — N(2); Y(2); M(4) S(2); RG(1) F(2) Y(2) A(1); H(2) D(1) GI Drom IGHV1S A(4) G(4) F(4) T(4) F(4)S(4) S(4) — — Y(4) W(3); M(4) Y(3); — (20, 22, 23, 24) Y(1) S(1) Family3 Hu GL A(16) G(16) F(16) T(16) F(16) S(13); S(11); — — Y(15); A(6);M(15); H(9); R(11); D(3) D(4); H(1) G(3); T(1) S(5); K(5) N(1) Y(2);N(2) W(2); S(1); E(1); T(1) GI Drom IGHV1S A(16) G(15); F(16) T(16)F(14); S(16) S(16) — — Y(15); W(7); M(13); Y(8); — (21, 25-39) A(1)S(1); C(1) A(4); I(2); S(8) Y(1) D(3); V(1) Y(1); C(1) Family 1 Hu GLA(6); G(7) Y(5); T(7) F(7) T(5); S(6); — — Y(7) A(4); I(4); S(4); R(4);V(1) G(2) S(2) D(1) G(2); M(3) N(2); T(1); -(1) Y(1) H(1) GI LlamaIGHV1S6 A(1) G(1) F(1) T(1) F(1) S(1) S(1) — — S(1) A(1) M(1) S(1) A(1)Family 1 Hu GL A(6); G(7) Y(5); T(7) F(7) T(5); S(6); — — Y(7) A(4);I(4); S(4); R(4); V(1) G(2) S(2) D(1) G(2); M(3) N(2); T(1); -(1) Y(1)H(1) Asterisks indicate key residues important for canonical folds.

TABLE 2(B) Comparison of H2 sequences for dromedary and llama germlineVH with human germlines; numbering according to Kabat et al. (Sequencesof Proteins of Immunological Interest, 5^(th) ed. (1991)). H2: 1 residueno. 5 5 5 5 5 5 5 5 5 5 5 5 7 0 1 2 2a 2b 2c 3 4 5 6 7 8 1 * * GI DromIGHV1S G(17); (18); Y(16); — — — S(19) D(18); G(19) S(18); T(19) Y(17);R(18); (1-19) A(2) N(1) N(2); R(1) G(1) N(1); Q(1) H(1) Family 3 Hu GLV(2); I(4) Y(2); — — — S(2); G(3); G(4) S(2); T4 Y(4) R(4) A(2) G(2)T(2) A(1) G(1); D(1) H2: 3 residue no. 5 5 5 5 5 5 5 5 5 5 5 5 7 0 1 22a 2b 2c 3 4 5 6 7 8 1 * * GI Drom IGHV1S T(3);G(1) I(4) N(4) S(4) — —G(3); G(4) G(3); S(3); T4 Y(4) R(4) (20, 22, 23, 24) D(1) S(1) N(1)Family 3 Hu GL V(5); I(15); S(12); Y(5); — — D(8); G(13); S(10); S(6);T(6); Y(12); R(16) Y(4); S(1) N(2); S(5); S(4); S(3) G(6) N(5); K(6)G(2); G(2); W(1); W(3); N(4) T(3); I(4) T(1); A(2); K(1) G(2); Y(1);N(1) R(1); Q(1) E(1) N(1); L (1) H2: 3 residue no. 5 5 5 5 5 5 5 5 5 5 55 7 0 1 2 2a 2b 2c 3 4 5 6 7 8 1 * * GI Drom IGHV1S A(9); I(16) N(10);S(10); — — G(14); G(16) G(16) S(16) T(16) Y(16) R(8); (21, 25-39) S(4);Y(6) T(6) A(2) K(4); T(2); Q(4) G(1) Family 1 Hu GL W(4); I(6); S(2);P(3); — — Y(2); N(2); G(7) N(4); T(3); N(4); A(3); R(1); V(1) N(2);T(2); N(2); T(2); T(1); P(2); T(2); L(2); G(1); I(2); A(2) I(2); L(1);I(1); A(2) I(1) T(2) L(1) D(1) E(1) F(1); E(1) D(1) H2: 2 residue no. 55 5 5 5 5 5 5 5 5 5 5 7 0 1 2 2a 2b 2c 3 4 5 6 7 8 1 * * GI LlamaIGHV1S6 S(1) I(1) Y(1) S(1) — — Y(1) S(1) S(1) N(1) T(1) Y(1) T(1)Family 1 Hu Gl W(4); I(6); S(2); P(3); — — Y(2); N(2); G(7) N(4); T(3);N(4); A(3); R(1); V(1) N(2); T(2); N(2); T(2); T(1); P(2); T(2); L(2);G(1); I(2); A(2) I(2); L(1); I(1); A(2) I(2) T(2) L(1) D(1) E(1) F(1);E(1) D(1) Asterisks indicate key residues important for canonical folds.

TABLE 3 Sites of key residues for determining canonical folds insomatically mutated dromedary VLambda sequences; numbering according toKabat et al. (Sequences of Proteins of Immunological Interest, 5^(th)ed. (1991)) and key residues proposed by Morea and colleagues (Morea etal., Methods 2000). Closest Canonical hu GL L1(λ) key residues L2(λ) keyresidues Sequence structure family 2 25 29 30 33 71 48 64 Som Mut DronVL3-1 L1: 11 (or 2) (IGLV3-1*01) — G(5) — F(4); T(2); T(4); (CamvI8, 18,19, 20, 23) L(1) A(1); A(1) V(1); E(1) Hu GI Vλ family 3 — G(10) — L(6);A(6); A(5); I(3); V(3); T(3); M(1) E(1) V(2) L1: 2 (Morea) — G — I V ASom Mut Dron VL3-12/32 L1: 11 (or 2) (IGLV3-1*01) — G(1) — L(1) A(1)I(1) (CanvI11) Hu GI Vλ family 3 — G(10) — L(6); A(6); A(5); I(3); V(3);T(3); M(1) E(1) V(2) L1: 2 (Morea) — G — I V A Som Mut Dron VL1-40 L1:14 (or 6) (IGLV1-40*01) S(1) G(1) N(1) — V(1) A(1) (CamvI44) Hu GI Vλfamily S(2) G(2) N(2) — V(2) A(2) L1: λ6 (Marin) S G N — V A Som MutDron VL2-18 L1: 14 (or 6) (IGLV2-18*02) S(11); G(12) D(12) — V(12)A(11); (CamvI5, 17, 30-33, 36, 52, 57, A(1) V(1) 59, 60, 65) Hu GI Vλfamily 2 S(6) G(6) D(6) — V(6) A(6) L1: λ6 (Marin) S G N — V A Som MutDron VL3-1 L2: 7 (IGLV3-1*01) I(4); G(3); (CamvI8, 18 19, 20, 23) L(1)A(2) Hu GI Vλ family 3 I(10) G(10) L2 1 (Morea) I; V G Som Mut DronVL3-12/32 L2: 7 (IGLV3-1*01) (1) G(1) (CanvI11) Hu GI Vλ family 3 I(10)G(10) L2 1 (Morea) I; V G Som Mut Dron VL1-40 L2: 7 (IGLV1-40*01) (1)E(1) (CamvI44) Hu GI Vλ family (2) G(2) L2 1 (Morea) I; V G Som Mut DronVL2-18 L2: 7 (IGLV2-18*02) I(12) G(11); (CamvI5, 17, 30-33, 36, 52, 57,59, S(1) 60, 65) Hu GI Vλ family 2 (6) G(6) L2 6 (Morea) I; V G

TABLE 4(A) Comparison of L1 sequences of dromedary VLambda with humangermlines; numbering according to Kabat et al. (Sequences of Proteins ofImmunological Interest, 5^(th) ed. (1991)). L1:11 residue no. 3 3 7 0 22 2 2 0 3 3 3 3 3 3 1 2 6 7 8 9 * 0a 0b 0c 1 2 * * Som Mut Drom VL3-1 —— G(4); N(3); — F(4); G(5) S(4); Y(4); — Y(4); T(2); A(5) (Camv18, 18,19, 20, 23) D(1) D(1); L(1) D(1) K(1) K(1) A(1); I(1) V(1); F(1) Family3 Hu G1 Vλ family 3 — — D(6); N(3); — L(6); G(5); S(4); K(6); — Y(7);A(6); A(3); N(3); A(3); I(3); K(3); Y(1) A(1); V(3); T(3); E(1) S(2);M(1) D(1); N(1); S(1); E(1) V(2); K(1); E(1) E(1) Q(1); N(1) V(1) G(1)S(1) L1:11 residue no. 3 7 0 2 2 2 2 0 3 3 3 3 3 3 1 2 6 7 8 9 * 0a 0b0c 1 2 3 * Som Mut Drom VL3- — — S(1) L(1) — R(1) N(1) Y(1) Y(19); —A(1) A(1) A(1) 12/32 (Camv111) C(1) Family 3 Hu G1 Vλ family 3 — — C(6);N(3); — L(6); G(5); S(4); K(6); — Y(7); A(6); A(3); N(3); A(3); I(3);P(3); K(3); Y(1); A(1); V(3); T(3); E(1) S(2); M(1) R(1); D(1); N(1);S(1); E(1) V(2) K(1); E(1) E(1); Q(1); N(1) V(1) G(1) S(1) L1:14 residueno. 0 2 3 7 2 2 2 2 9 3 3 3 3 3 3 3 1 * 6 7 8 * 0 0a 0b 0c 1 2 * * SomMut Drom VL1- S(1) S(1) S(1) S(1) N(1) I(1) G(1) G(1) G(1) S(1) G(1)V(1) A(1) 40 (Camv144) Family 1 Hu G1 Vλ family 1 G(2) E(2) S(2) S(2)N(2) I(2) G(2) A(2) G(2) Y(2) D(1); V(2) A(2) V(1) L1:14 residue no. 0 23 7 2 2 2 2 9 3 3 3 3 3 3 3 1 * 6 7 8 * 0 0a 0b 0c 1 2 * * Som Mut DromVL2- S(1); T(12) S(10); N(3); D(11); V(12) G(12) G(5); Y(2) N(11); Y(12)V(12) A(1); 18 (Camv15, 17,30- A(1) F(2) D(3) G(1) R(4); A(1) N(1)33,36,52,57,59,60,65) K(2); A(1) Family 2 Hu G1 Vλ family 2 S(6) T(6)S(6) S(6) D(6) V(6) G(6) G(3); Y(6) N(5); Y(3); V(6) A(6) S(2); D(1)F(1); D(1) L(1); Y(1) Asterisks indicate key residues important forcanonical folds.

TABLE 4(B) Comparison of L2 sequences of dromedary VLambda with humangermlines; numbering according to Kabat et al. (Sequences of Proteins ofImmunological Interest, 5^(th) ed. (1991)). L2: 7 residue no. 4 6 8 4 55 5 5 4 * 9 0 1 2 3 * Som Mut Drom VL3-1 I(4); Y(5) K(3); D(4); S(2);A(1); G(3); (Camv8, 18, 19, 20, 23) L(1) R(1); N(1) T(2); L(1); A(2)G(1) D(1) E(1); N(1); S(1) Family 3 Hu GI Vλ family 3 I(10) Y(10) E(2);D(8); S(9); E(3); G(10) K(2); K(1); N(1) N(3); D(1); S(1) K(2); Q(1);D(2) R(1); S(1); G(1); Y(1) Som Mut Drom VL3-12/32 I(1) Y(1) N(′) D(1)N(1) N(1) G(1) (CamvI11) Family 3 Hu GI Vλ family 3 I(10) Y(10) E(2);D(8); S(9); E(3); G(10) K(2); K(1); N(1) N(3); D(1); S(1) K(2); Q(1);D(2) R(1); S(1); G(1); Y(1) Som Mut Drom VL1-40 I(1) Y(1) G(′) N(1) S(1)N(1) E(1) (CamvI44) Family 1 Hu GI Vλ family 1 I(2) Y(2) G(2) N(2) S(2)N(2) G(2) Som Mut Drom VL2-18 I(12) Y(12) Q(11); V(9); N(10); K(12)G(11); (CamvI5, 17, 30-33, 36, 52, 57, D(1) I(2); S(1); S(1) 59, 60, 65)D(1) D(1) Family 2 Hu GI Vλ family 2 I(6) Y(6) E(4); V(6) S(5); K(3);G(6) D(′); N(1) N(2); N(1) T(1) Asterisks indicate key residuesimportant for canonical folds.

TABLE 5 Sites of key residues for determining canonical folds insomatically mutated dromedary VKappa sequences; numbering according toKabat et al. (Sequences of Proteins of Immunological Interest, 5^(th)ed. (1991)) and key residues proposed by Morea and colleagues (Morea etal., Methods 2000). Canonical Closest L1(κ) key residues L2(κ) keyresidues Sequence structure hu GL family 2 25 29 30e 33 41 48 64 Som MutDrom VK2-10 K1: 3 IGKV2-40 I(7) S(7) V(6); Q(7) L(7) F(7) (Kp1, 3, 6, 7,10, 20, 48) (2_1 or 011 */01) L(1) Hu GL VK family 2 I(1) S(1) L(1) G(1)L(1) F(1) K1: 0 I O L; V C; Q; G L T (Morea) Som Mut Drom VK2-40 K2: 1IGKV2-40 I(7) G(7) (Kp1, 3, 6, 7, 10, 20, 48) (2_1 or 011 */01) HuGL VKfamily 2 I(1) G(1) H2: 1 I; V G (Morea)

TABLE 6 Comparison of (A) L1 and (B) L2 sequences of dromedary VKappawith human germline; numbering according to Kabat et al. (Sequences ofProteins of Immunological Interest, 5^(th) ed. (1991)). L1(κ): 11residue no. 2 2 3 7 2 5 2 2 2 9 3 3 3 3 3 3 3 3 3 3 1 * * 6 7 8 * 0 0a0b 0c 0d 0e 0f 1 2 * * Som Mut I S S Q S V F S S S N Q K S Y L F Drom(7) (7) (7) (7) (5); (6); (3); (5); (4); (7) (4); (7) (6); (7) (3); (7)(7) VK2-40 N L L D D S R L (Kp1, (1); (1) (2); (1); (1); (2); (1) (2);3, 6, H I A T R Q 7, 10, (1) (1); (1) (1); (1) (1); 20, 48) V V F (1);(1) (1) Fam- Hu GI I S S Q S L L D S D D G N T Y L F ily VK (1) (1) (1)(1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) 2 family 2 (011*/01) (B) L2(κ): 11 residue no. 4 6 8 4 5 5 5 4 * 9 0 1 2 * Som Mut I(7)Y(7) Y(7) A(7) S(7) G(7) Drom VK2-40 (Kp1, 3, 6, 7, 10, 20, 48) Fam- HuGI VK I(1) Y(1) T(1) I(1) S(1) G(1) ily family 2 2 (011 */01) Asterisksindicate key residues important for canonical folds.

Example 14 Sequence Analysis of Somatically Mutated VH, VK and VL fromLlama

From four llamas peripheral blood lymphocytes were isolated, RNAextracted and random primed cDNA synthesized as described before (deHeard et al, JBC 1999). Amplification of VHCH1, VLCL and VKCK wasperformed and the amplicons were cloned in vector pCB3 yielding heavychain or light chain libraries. After screening of clones with PCR tocheck for the presence of the antibody domain insert, individual cloneswere grown and plasmid DNA was purified for sequence analysis.

Section 14.A shows the lambda light chain variable regions grouped intofamilies according to the closest human germline analogue with the sameCDR1 and CDR2 length and presumable having the same canonical foldcombination. The lambda germlines most frequently used in humans, i.e.VL1, VL2 and VL3, are also often found in the analyzed llama sequencesand in addition VL4, VL5, VL6 (not shown in section 14.A) and VL8,meaning that 7 out of the 10 lambda families as found in humans are usedas well in the llama. Section 14.B shows two of the three kappa lightchain variable regions (i.e. VK1, and VK4; VK2 is not shown), whichoccur in about 50% of kappa containing human antibodies. Members of theVK3 family, which is used most frequently (50%) in human antibodies,were not identified, but it can well be that the used primers foramplification are responsible for this and that these have to beadapted. Section 14.0 shows the alignment of the VH sequences revealinga high sequence homology to the most often used human VH3 segment(occurs 34% in human antibodies) and VH1 (17%). It must be noted thatthe recent publication of Achour et al (J Immunol 2008) mentions thepresence of a VH2 family in the germline of Alpaca which is most closelyrelated to the human VH 4 family (see example 10.10).

Overall it can be concluded that camelids use a high diversity of heavychain and light chain families similar to what is found in the humanimmune system, meaning that by active immunization with human diseasetargets an excellent choice of lead antibodies can be expected with ahigh sequence homology to human antibodies, which therefore can beeasily engineered for therapeutic applications.

14.(A) VLAMBDA (SEQ ID NOS: 111-138) SEQ ID NOS: 139-149)   FR1              CDR1             FR2         CDR2               2             3             4          5 1234567891234567890123 45678901abc234 567890123456789 01abcdeHuVL1-1(3/4/5) QSVLTQPPSVSEAPRQRVTISC SGSSSNIGNN AVN WYQQLPGKAPKLLIY YDLAMBDA#14 QPVLTQPPSVSGSPGQKFTISC TGSSSNIGNN YVN WYQHLPGTAPRLLIY SNLAMBDA#16 QSALTQPPSVSGSPGQKFTISC TGSSSNIGDN YVN WYQHLPGTAPKLLIY SNLAMBDA#46 NFMLTQPPSVSGSPGQKFTISC TGSSSDIGNN YVN WYQHLPGTAPKLLIY STLAMBDA#45 NFMLTQPPSVSGSPGQKFTISC TGSSSNIGEN FVN WYQHLPGTAPKLLIY STLAMBDA#15 NFMLTQPPSVSGSPGQKFTISC TGSNNNIGNN YVN WYQHLPGTAPKLLIY SN CDR2              FR3                     CDR3          FR4         6           7         8          923456 789012345678ab90123456789012345678 9012345abcde HuVL1-1(3/4/5)DLLPS GVSDRFSGSKSG  TSASLAISGLQSEDEADYYC AAWDDSLNG LAMBDA#14NNRAS GVPDRFSGSKSG  SSASLTITGLQAEDEAEYYC SSWDDSLSGTV  FGGGTHLTVLLAMBDA#16SNRAS GVPDRFSGSKSG  SSASLTITGLQAEDEADYYC SSWDDSLSGHPV FGGGTKLTVLLAMBDA#46DKRAS GVPDRFSGSKSG  SSASLTITGLQAEDEADYYC SSWDDNLGTYV  FGGGTSVTVLLAMBDA#45DKRAS GVPDRFSGSKSG  SSASLTITGLQAEDEADYYC SSWDDNLGTYV  FGGGTSVTVLLAMBDA#15NYRAS GVPDRFSGSKSG  SSASLTITGLQAEDEADYYC SSWDESLSGRYV FGGGTKLAVL   FR1              CDR1             FR2         CDR2               2             3             4          51234567891234567890123 45678901abc234 567890123456789 01abcde HuVL1-2QSVLTQPPSVSGAPGQRVTISC TGSSSNIGAGYDVH WYQQLPGTAPKLLIY GN LAMBDA#47QSALTQPPSVSGTLGKTVTISC AGTSNDIGRYNYVA WYQQLPGTAPKLLIY AV LAMBDA#18QSALTQPPSVSGTLGKTLTISC AGTSSDVGYGNYVS WYQQLPGTAPKLLIY RV LAMBDA#32QSALTQPPSVSGTLGKTVTISC AGTRTDVGYGDYVS WYQHVPNTAPRLLIY AV LAMBDA#28QAVLTQPPSVSGTLGKAVTISC AGTGSDVGYGDYVS WYQQLPDTAPKLLVY AV LAMBDA#29NFMLTQPPSVSGSPGKTVTISC AGTSSDVGYGNYVS WYQQLPGMAPKLLLY NI LAMBDA#27QSALTQPPSVSGTLGKTVTISC AGTNSDIGDYNFVS WYQHLPGMAPKLLIY DV LAMBDA#17QAGLTQPPSVSGSLGKTITISC AGTRNDIGGHGYVS WYQQLPGTAPKLLIY KI LAMBDA#4SSELTQPPSVSGTLGKTVTISC AGTSNDIGAHNYVS WYQQLPGTAPKLLIN KV LAMBDA#7QPVLTQPPSVSGSPGKTVTISC AGTSSDIGYGNYVS WYQLLPGTAPKLLIY DV LAMBDA#8QSALTQPPSVSGSLGKTVTISC AGTIGDIGAGNYVS WYRQTPGTAPKLLLY EV LAMBDA#5QPVLTQPPSVSGSLGKTVTISC AGTWSDIGGYNYIS WYRQLPGTAPRLLIY EV CDR2              FR3                     CDR3          FR4         6           7         8          923456 789012345678ab90123456789012345678 9012345abcde HuVL1-2SNRPS GVPDRFSGSKSG  TSASLAITGLQAEDEADYYC QSYDSSLSG LAMBDA#47SYRAS GIPDRFSGSKSG  NTASLTISGLQSGDEADYYC VSYRSGGTNV   FGGGTHLTVLLAMBDA#18SYRPS GIPDRFSGSKSG  NTASLTISGLQSEDEADYYC TSYTYKGGGTAV FGGGTHLTVLLAMBDA#32SARAS GIPSRFSGSKSG  NTASLTISGLQSEDEADYYC ASYRDGNYAV   FGGGTHLTVLLAMBDA#28NTRAS GIPDRFSGSRSG  NTASLTISGLQSEDEGDYYC ASYRSYNNYV   FGGGTHLTVLLAMBDA#29NKRAS GIADRFSGSKSG  NTASLTISGLQSEDEAVYYC ASYRSGNNYV   FGGGTELTVLLAMBDA#27NKRAS GIADRFSGSKSG  NTASLTISGLQSEDEADYYC ASYRSSNNYV   FGGGTKLTVLLAMBDA#17NTRAS GIPDRFSGSKSG  NTASLTISGLQSEDEADYFC VADINGDTNV   FGGGTHLTVLLAMBDA#4STRAS GIPDRFSGSKSG  NTASLTISGLQSEDEADYYC AAYRTGDARI   FGGGTHLTVLLAMBDA#7NKRPS GIPDRFSGSKSG  NQAYLTISGLQSEDEADYYC VSYREPNNFV   SGGGTHLVVLLAMBDA#8NKRTS GIPDRFAGSRSG  NTASLIISGLQAEDEADYYC ASYRIGSRGV   FGGGTHLTVLLAMBDA#5DKRAP GIPDRFSGSKSG  TTASLVISGLQSEDEADYYC ASYKSSENAV   FGGGTHLTVV          FR1              CDR1             FR2        CDR2                  2          3             4          51234567891234567890123 45678901abc234 567890123456789 01abcde  HuVL2-1QSALTQPPSASGSPGQSVTISC TGTSSDVGSYNYVS WYQQHPGKAPKLMIY EV LAMBDA#1QSVLTQPPSVSGTLGKTLTISC AGTSSDVGYGNYVS WYQQLPGTAPKLLIY RV LAMBDA#8QSVLTQPPSVSGTLGKTVTISC AGTSSDVGYGNYVS WYQKLPGTAPKLLIY AV LAMBDA#5LPVLTQPPSVSGTLGKSLTISC AGTSSDVGNGNYVS WYQQLPGTAPKLLIY RV LAMBDA#11QPVLTQPPSVSGTLGKTVTISC AGTSTDIGGYNYVS WYQQVPGTAPKLLIY EV CDR2               FR3                     CDR3         FR4         6           7         8          923456 789012345678ab90123456789012345678 9012345abcde  HuVL2-1SKRPS GVPDRFSGSKSG  NTASLTVSGLQAEDEADYYC SSYAGSNNF LAMBDA#1STRAS GIPDRFSGSKSG  NTASLTISGLQSEDEADYYC SSYRSTGTAV   FGGGTHLSVLLAMBDA#8SYRAS GIPDRFSGSKSG  NTASLTISGLQSEDEADYYC ASYRDSNNAV   FGGGTHLTALLAMBDA#5TSRAS GVPDRFSGSKSG  NTASLTISGLQPEDEADYYC ASYKRGGTSV   FGGGTHLTVLLAMBDA#11NKRPS GIPDRFSGSKSG  NTASLTISGLQSEDEADYYC ASYRSSNNVV   FGGGTHLTVL          FR1              CDR1             FR2        CDR2                  2          3             4          51234567891234567890123 45678901abc234 567890123456789 01abcde HuVL3-2SYELTQPLSVSVALGQTARITC GGNNIGSK   NVH WYQQKPGQAPVLVIY RD LAMBDA#13LPVLTQPSALSVTLGQTAKITC QGGSLGSS   YAH WYQQKPGPAPVLVIY DD LAMBDA#40QAVLTQPSAVSVSLGQTARLTC QGDNVETA   GTS WYRQKPGQAPSLIIY GD LAMBDA#2QAGLTQPSAVSVSLGQTARITC RGDSLERY   GAN WYQQKPGQARVQVIY GD LAMBDA#14QSALTQPSAVSVSLGQTAEITC RGRNFESG   FPH WYRQKPGQSPELVMF IV CDR2               FR3                     CDR3         FR4         6           7         8          923456 789012345678ab90123456789012345678 9012345abcde  HuVL3-2SNRPS GIPERFSGSNSG  NTATLTISRAQAGDEADYYC QVWDSSTA LAMBDA#13ANRPS GIPERFSGSRSG  GTATLTISGAQAEDEGDYYC QSVDNSGNVV   FGGGTHLTVLLAMBDA#40SSRPS EISERFSASTSG  NTATLTITGAQAEDEADYYC LSADSDLDSV   FGGGTLLTVLLAMBDA#2DIRPS GIPERFSGSRLG  GTATLTISGAQAEDEADYYC QSSDSSGYMND  FSSRTHLTVLLAMBDA#14NNRWS GIPDRFSGTRSG  DAATLTITGVQAEDEADYYC QMWDGEGAV    FGGGTHLTVL          FR1              CDR1             FR2        CDR2                  2          3             4          51234567891234567890123 45678901abc234 567890123456789 01abcde HuVL4-1LPVLTQPPSASALLGASIKLTC TLSSEHSTY  TIE WYQQRPGRSPQYIMK VKSDGS LAMBDA#10QSVLTQPPSASASLGASAKLTC TLSSGYSSY  NVD WYQQVPGKSPWFLMR VGSSGV CDR2               FR3                     CDR3         FR4         6           7         8          923456 789012345678ab90123456789012345678 9012345abcde  HuVL4-1HSKGD GIPDRFMGSSSG  ADRYLTFSNLQSDDEAEYHC GESHTIDGQVG LAMBDA#10GSKGS GVSDRFSGSSSG  LERYLTIQNVQEEDEAEYIC GADHASSMYT   FGGGTHLTVL          FR1              CDR1             FR2        CDR2                  2          3             4          51234567891234567890123 45678901abc234 567890123456789 01abcde HuVL5-1QPVLTQPPSSSASPGESARLTC TLPSDINVGSYNIY WYQQKPGSPPRYLLY YYSDSD LAMBDA#3LPVLTQPPSLSASPGASARLTC SLNSGTIVGGYHIN WYQQKAGSPPRYLLR FYSDSN LAMBDA#7LPVLTQPPSLSASPGASARLTC VLSSGTVVGGYHIN WYQQKPGSPPRYLLR FYSDSS LAMBDA#9LPVLNQPPSLSASPGESARLTC SLSSETIVGGYQIA WYQQTAGSPPRYLLR FYSDSN LAMBDA#26LPVLTQPPSLSASPGSSVRLTC TLSSGKSVGMYDIS WYQQKAGSPPRYLLY YYSDTS LAMBDA#12QSVLTQPPSLSASPGSSVRLTC TLSSANSVDNYYIS WYQQKPGSPPRYLLY YYSDSY CDR2               FR3                     CDR3         FR4         6           7         8          923456 789012345678ab90123456789012345678 9012345abcde  HuVL5-1KGQGS GVPSRFSGSKDASANTGILLISGLQSEDEADYYC MIWPSNAS LAMBDA#3KHQGS GVPSRFSGSKDASANAGLLLISGLQVEDEADYYC GIYDSNTGTYV  FGGGTKLTVLLAMBDA#7KQQGS GVPSRFSGSKDASANAGLLLISGLQPEDEADYYC GTYHSNTGTYV  FGGGTKLTVLLAMBDA#9KHQGS GVPSRFSGSKDASANAGILFISGLQPEDEADYYC GIYHYNSDTYV  FGGGTRLTVLLAMBDA#26NHQGS GVPSRFSGSKDASANAGLLLISGLQPEDEADYYC ATGDRSSNPHV  FGGGTKLTVLLAMBDA#12MQRDS GLPDRFSVSKDASTNAGLLLISGLQPEDEADYYC ASGDRNSNPHSV FGGGTHLTVL          FR1              CDR1             FR2        CDR2                  2          3             4          51234567891234567890123 45678901abc234 567890123456789 01abcde HuVL8-1QTVVTQEPSLTVSPGGTVTLTC ASSTGAVTSGYYPN WFQQKPGQAPRALIY ST LAMBDA#42QAVVSQEPSLSVSPGGTVTLTC GLSSGSVTTSNYPG WFQQTPGQAPRTLIY ST LAMBDA#31QTVVTQEPSLSVSPGGTVTLTC GLTSGSVTASNLPG WFQQTPGQAPRTLIF DT CDR2               FR3                     CDR3         FR4         6           7         8          923456 789012345678ab90123456789012345678 9012345abcde  HuVL8-1SNKHS WTPARFSGSLLG  GKAALTLSGVQPEDEAEYYC LLYYGGAQ LAMBDA#42SSRHS GVPSRFSGSISG  NKAALTITGAQPEDEADYYC ALDIGSYTV     FGGGTKLTVLLAMBDA#31IYHHS GVPSRFSGSIAG  NKATLTITGAQPEDEGDYFC VLWMDRIEAGSIM FGGGTHLSVV

14. (B) VKA.PPA (SEQ ID NOS: 150-186)                                L1                         L2         FR1                   CDR1             FR2       CDR2         1         2          3                4          5 12345678901234567890123 45678901abcdef234 567890123456789 0123 HuVK1-1DIQMTQSPSSLSASVGDRVTITC RASQSI      SSYLN WYQQKPGKAPKLLIY AASS KAPPA#39DIQLTQSPSSLSASLGDRVTITC QASQSI      STELS WYQQKPGQTPKLLIY GASR KAPPA#22DIQLTQSPSSLSASLGDRVTITC QASQSI      STELS WYQQKPGQTPKLLIY DRSR KAPPA#24DIVMTQTPSSLSASLGDRVTITC QASQSI      NTELS WYQQKPEQPPKLLIY AASR KAPPA#21DIVMTQSPSSLSASLGDRVTITC QATQSI      NTELS WYQQKPGQSPKLLIY EASR KAPPA#23DIVMTQTPSSLSASLGDRVTITC QASQSI      STELA WYQQKPGQTPKLLIY GASK KAPPA#7AIQMTQSPSSLSASLGDRVTITC QASQSI      STELS WYQQKPGQTPKLLIY GASR KAPPA#19DIQLTQSPSSLSASLGDRVTITC QASQSI      STELS WYQQKPGQTPKLLIY GASR KAPPA#25bDIVMTQTPPSLSASLGDRVTITC QASQSI      RNELA WYQQKPGQTPKLLIY GASR I-_8AIQMTQSPSSLSASLGDRVTITC QASQSI      SSYLA WYQQKPGQAPKLLIY GAST KAPPA#43AIQMTQSPSSLSASLGDRVTITC QASQSI      SSYLA WYQQKPGQAPKLLIY GASR KAPPA#44VIQMTQSPSSLSASLGDRVTITC QASQSI      SNYLA WYQQKPGQAPKLVIY GASR KAPPA#20bDIQLTQSPSSLSASLGDIVTITC QASQSI      TTELS WYQQKPGQTPKLLIY GAFR L2                                     L3CDR2               FR3                 CDR3       6         7         8          9456 78901234567890123456789012345678 9012345 HuVK1-1LQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQSYSTP KAPPA#39LQT GVPSRFSGSGSGTSFTLTISGLEAEDLATYYC LQDYSSPYS  FGSGTRLEIK KAPPA#22LQI GVPSRFSGSGSGTTYTLTISDLEAEDLATYYC LQDDSWPYS  FGSGTRLEIK KAPPA#24LQT GVPSRFSGSGSGTSFTLTISGLEAEDLATYYC LQDSDWPLT  FGQGTKVELK KAPPA#21LQT GVPSRFSGSGSGTSFTLTISGLEAEDLATYYC MADLDWPLV  FGQGTKVELK KAPPA#23LQT GVPSRFSGSGSGTSFTLTISGLEAEDLATYYC LQGYSSPLT  FGQGTEVDLK KAPPA#7LQT GVPSRFSGSGSGTSFTLTISGLESEDLATYYC LQDYSWPLT  FGQGTKVELT KAPPA#19LQT GVPSRFSGSGSGTSFTLTISGLEAEDLATYYC LQDYSWPRT  FGQGTKLEIK KAPPA#25bLQT GVPSRFSGSGSGTSFTLTISGLEAEDLATYYC LQDDSWPLT  FGQGTKVELK I-_8LQT GVPSRFSGSGSGTSFTLTISGLEAEDAGTYYC QQYYSIPVT  FGQGTKVELR KAPPA#43LQT GVPSRFSGSGSGTSFTLTISGLEAEDAGTYYC QLYGSRPS   FGQGTKVELK KAPPA#44LQT GVPSRFSGSGSGTSFTLTISGLEAEDAGTYYC QQYYSTYS   FGSGTRLEIK KAPPA#20bLQA GVPSRFSGSRSGTTFTLTISGLEAEDLATYYC LQDYSWPPYS FGSGTRLEIK                                L1                         L2         FR1                   CDR1             FR2       CDR2         1         2          3                4          5 12345678901234567890123 45678901abcdef234 567890123456789 0123 HuVK4-1DIVMTQSPDSLAVSLGERATINC KSSQSVLYSSNNKNYLA WYQQKPGQPPKLLIY WAST KAPPA#53ETTLTQSPSSVTASVGEKVTINC KSSQNVGSGSNQKSILN WIQQRPGQSPRLLIY YAST KAPPA#50EIVMTQSPSSVTASAGEKVTINC KSSQSVFQSSNQKNYLG WYQQRIGQSPRLLIN WAST KAPPA#20DIVMTQTPSSVTASIGEKVTINC KSSQSVLYSSNQKNYLT WYQQRLGQSPRLLIY WAST KAPPA#48ETTLTQSPSSVTASAGEKVTINC KSSQSVLLDSNQKNYLA WYQQRLGQSPRLLIY WAST KAPPA#55EIVLTQSPSSVTASAGEKVTINC KSSQSVLYSSDQKNYLA WYQQRPGQSPRLLIY WAST KAPPA#9ETTLTQSPSSVTASAGEKVTINC KSSQSVLYRSDQKNVLS WYQQRLGQSPRLLIY WAST KAPPA#51DIVMTQTPSSVTASAGEKVTINC KSSQSVLNNSDQKIYLA WYQQRLGQSPRLLIY WAST KAPPA#10DIVMTQSPGSVTASTGENITINC KSSQNVLLSSDQKNYLN WYQQRLGQSPRLLIY WAST KAPPA#54DIVMTQTPTSVTASAGEKVTINC KSSQSLLYSANQKVYLA WYQQRLGQSPRLLFR WTST KAPPA#49DIVMTQTPTSVTASAGEKVTINC KSSQSLLYSANQKVYLA WYQQRLGQSPRLLFR WTST KAPPA#52EIVMTQSPTSVTASVGEKVTINC KSSQSLLYSANQKVYLA WYQQRLGQSPRLLFY WTST KAPPA#13EIVLTQSPSSVTASVGEKVTINC KSSQSVKSGSNQITYLN WYQQTPGQSPRLLIY YAST KAPPA#11ETTLTQSPSSVTASVGEKVTINC KSSQSVVSGSNQKIYLN WYQQRPGQSPRLLIY YAST KAPPA#36DIVMTQTPSSVTASVGETVTIGC KSSQSVVSGSSQKSFLN WYQQRPGQSPRLLIY YAST KAPPA#25DIVMTQTPRSVTASVGEKVTINC KSSQSVLSGSNQKSYLN WYQTRPGQSPRLLIY YAST KAPPA#12DVVMTQSPSSVTASVGEKVTINC KSSQSVRSGSNEKSSLN WYQQRPGQSPRLLIY YAST KAPPA#34DVVMTQSPSSVTASVGEKVTIDC KSSQILVSGSDQKSYLS WYQQRPGQSPRLLIY YAST KAPPA#48bEIVMTQTPSSVTASVGEKVTINC KSSQSVVLASNQKTYLN WYQQRPGQSPRLLIY YAST KAPPA#21DVVMTQSPSSVTASVGEKVTINC KSSQSVVSGSNQKSYLN WYQQRPGQSPRLLIY YAST KAPPA#24EIVMTQSPSTVTASVGEKVTIKC KSSQSVVSGSNQKTYLN WYQQRPEQSPRLLMY YAAT KAPPA#22bDVVMTQSPSSVTASAGEKVTINC KSSQSLLWSDNKKNYLS WYQQRLGQSPRLLIY WAST KAPPA#45DIVLTQSPSSVTASAGEKVTITC ESSQSVLRSSNQRNYLN WYQQRLGQSPRLIIY WAST KAPPA#46EIVLTQSPSSVTASAGEKVTINC KSSQSVLYSSNQKNYLA WYQQRLGQSPRLLIY WAST L2                                     L3CDR2               FR3                 CDR3       6         7         8          9456 78901234567890123456789012345678 9012345 HuVK4-1RES GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC QQYYSTP KAPPA#53RDA GIPDRFSGSGSATDFTLTIRSVQPEDAAVYYC QQVNIAPYT FGSGTRLEIR KAPPA#50RES GVPDRFSGSGSTTDFTLTINPFQPEDAAVYYC QQGKSAPLT FGQGTKVELK KAPPA#20RES GVPDRFSGSGSLTTFTLTISSFQPEDAAVYFC QQGYSVPLT FGRGTKVELK KAPPA#48RQS GVPDRFSGSGSTTDFTLTISSFQPEDAAVYYC QQGITIPVT FGQGTKVELK KAPPA#55RES GVPDRFSGSGSTTDFTLTISSFQPEDAAVYYC QQGYSSPHS FGSGTRLEIK KAPPA#9RES GVPDRFSGSGSTTDFTLTISSFQPEDAAVYYC QQGYSRPYS FGNGTRLEIK KAPPA#51RES GVPDRFSGSGSTTDFTLTISSFQPEDAAVYYC QQEYSAPAS FGSGTRLEIK KAPPA#10RKS GIPDRFSGRGSTTDFTLTINSFQPEDAAVYYC QQGYSIPHT FGGGTRLEIK KAPPA#54RQP GIPDRFSGSGSTTDFTLTISRVQPEDAAVYYC QQAYARPHT FGSGTRLEIK KAPPA#49RQP GIPDRFSVSGSTTDFTLTISSVQPEDAAVYYC QQAYARPHT FGSGTRLEIK KAPPA#52RQS GIPDRFSGSGSTTSFTLTISGVQPEDAAVYYC QQAYTRPHT FGSGTRLEIR KAPPA#13QEL GIPDRFSGSGSTTDFTLTISSVQPEDAAVYYC QQAYSAPFS FGSGTRLEIK KAPPA#11QES GIPDRFSGSGSTTDFTLTISSVQPEDAAVYYC QQGASAPVS FGSGTRLEIK KAPPA#36LEL GIPDRFSGSGSTTDFTLTISSVQPEDAAVYYC QQAYSTPST FGPGTKLEIR KAPPA#25QES GIPDRFSGSGSTTDFTLTISGVQPEDAAVYYC QQAYSAPAT FGQGTTVEVI KAPPA#12QES GIPDRFSGSGSTTDFTLTISSVQPEDAAVYYC QQAYSYPIT FGQGTKVELK KAPPA#34QKL GIPDRFSGSGSTTDFTLTISSVQPEDAAVYYC QQTYEAPYS FGNGTRLEIK KAPPA#48bQQL GIPDRFSGSGSTTDFTLTISSVQPEDAAVYYC QQALSAPYS FGSGTRLEIK KAPPA#21QEL GIPDRFSGSGSTTDFTLTISSVQPEDAAVYYC QQAYSTPYS FGSGTRLEIK KAPPA#24PEL GIPDRFSGSGSTTDFTLTINSVQPEDAAVYYC QQTYSPPN  FGSGTRLEIA KAPPA#22bRES GAPDRFSGSGSTTDFTLTISNFQPEDAAVYYC QQGYSIPIT FGQGTKVELS KAPPA#45RES GVPDRFSGSGSTTDFTLTISSFQPEDAAVYYC QQASSLPFT FGQGTKVELK KAPPA#46RES GVPDRFSGSGSTTDFTLTISSFQPEDAAVYYC QQYLSGVT  FGQGTKVELK

14. (C) VH (SEQ ID NOS: 187-226)                              H1                           H2            FR1                 CDR1        FR2              CDR2         1         2         3             4          5            6123456789012345678901234567890 1ab2345 67890123456789 012abc3456789012345VH1(1-02)QVQLVQSGAEVKKPGASVKVSCKASGYTFT G--YYMH WVRQAPGQGLEWMG WINP--NSGGTNYAQKFQG1C2EVQLVQSGAELRNPGASVKVSCKASGYTFT S--YYID WVRQAPGQGLEWMG RIDP--EDGDTKYAPKFQG1G5EVQLVQSGAELRNPGASVKVSCKASGYTFT S--YYIE WVRQAPGQGLEWMG RIDP--EDGGTKYAQKFQGVH3(3-23)EVQLLESGGGLVQPGGSLRLSCAASGFTFS S--YAMS WVRQAPGKGLEWVS AISG--SGGSTYYADSVKG5B12EVQLVESGGGLVQPGGSLRLSCTTSGFTFE D--YPMN WVRQAPGKGLEWVS VISR--NGGSTYYAESMKG1G4EVQLVESGGGLVQPGGSLRLSCAASGFTFD D--YGMS WVRQAPGKGLEWVS GITW--NGGTTNYADSVKG5G7EVQLVESGGGLVQPGGSLRVSCAASGFTFS T--YYMS WVRQAPGKGLEWVS GINT--GGDSTYYADSVMGVH_99EVQLVESGGGLVQPGGSLRLSCSASGFRFS T--YAMT WVRQAPGKGLVWVS TVDA--SGATTSYAESVKGVH_76EVQLVESGGGLVQSGGSLRLSCAASGFTFS D--YAMS WVRQAPGKGLEWVS SINN--GGWSTRYADSVKGVH_86EVQLVQSGGGLVQPGGSLRLSCAASGFTFS R--YSMS WVRQAPGQGLEWVS YIDS--DGATTTYADSVKGVH_98EVQLVESGGGLVQPGGSLRLSCAASGFTFS I--YGMS WVRQAPGKGLEWVS VINS--GGDSTSYADSVKGVH_89EVQLVESGGGLVQPGGSLRLSCSASGLTAS N--TAMA WVRQVPGKQLEWVS DINS--LGNNIFYSKSVKGVH_82EVQLMQSGGGLAQPGGSLRLSCAASGFTLS N--HWMY WVRQAPGKGLEWVS AISS--SGSSTYYIDSVKGVH_68EVQLVQSGGGLVQPGGSLRLSCAASGFAFS S--SDMS WVRQAPTKGLEWVS GINS--GGGSTYYGESMKGVH_73EVQLVQSGGDLVQPGGSLRLSCAASGFAFS S--YHIS WVRQAPGKGLEWVS IIGR--WGADIYYADSVKGVH_107EVQLVESGGGLVQPGGSLRLSCVGSGITFS K--YAMS WVRQAPGKGLEWVS NIDA--NSELTTYEDTVKGVH_94EVQLVQSGGGLVQPGGSLRLSCAASGFTFS S--YYMN WVRQPPGKGLEWLS VISS--SGGNTKYSDSVKGVH_90EVQLVQSGGGLVQPGGSLRLSCAASGFTFD D--YGKT WVRQAPGKGLEWVS SIYI--FVGNTYYADSVKGVH_77EVQLVQSGGGLVQPGGSLRLSCAASGFTFS S--YAMS WVRQAPGKGLEWVS TISS--GGASTTYADSVKGVH_74EVQLVQSGGGLVQPGGSLRLSCAASGFAFS D--YDMS WVRQAPGKGLEWVS IHVS--GDGRIFYADSMKGVH_67EVQLVQSGGGLVQPGGSLRLSCAASGFRFT D--YYMG WIRQTPGKGLEWVS SIYS--LGDPTYYADSVKGVH_79EVQLVESGGGLVQPGGSLRLSCAASGFAFS R--YWMY WVRQAPGKGLEWVS GMTT--GSDYIYSAVSVKGVH_75EVQLVGVWGRLGAPGGSLRLSCAASGFPFS I--YFMS WFRQRPEKGARMVS DIDK--SGGRTTYAPSVKGVH_110EVQLVQSGGDLVQPGGSLRVSCAVSGFTFI Y--YGMS WVRQSPGKGLEWIS TISN--GGSTANYADSVKGVH_109EVQLVESGGGLVQPGGSLRLSCTASGFTFS S--YGIS WVRQAPGKGLEWVS SVTG--DGLSTTAIDSVKGVH_103EVQLVQSGGGLVQPGGSLRLSCAASGFTFR S--YYMN WVRQAPGKGLEWVS VSSS--GGGTTYYADSVKGVH_84EVQLVESGGGLVQPGGSLRLSCAASGFTFS S--YAMS WVRQAPGKGLEWVS GINS--GGGSTSYADSVKGVH_113EVQLVESGGGLVQPGRSLRLSCAAAGFTFS T--YWMY WIRQAPGKGLEWVA TITS--LGGSQWYVDSVKGVH_87EVQLVESGGGLVKPGGSLRLSCAASGFTFS S--AYMN WVRQAPGKGLEWVS GLTN--YGSTSYYADSVKGVH_105EVQLVESGGGLVQPGGSLRLSCAASGFTFS N--YWMY WVRQAPGKGLEWVS SIDT--SGGITMYADSVKGVH_101EVQLVESGGGLVQPGVSLRLSCTTSGFTFS T--QGMN WVRQPPEKGLEWVS GIDS--RGNTTNYADSVKGVH_108EVQLVQSGGGLVQPGGSLRLSCAASGFTFS S--YWMY WVRQAPGKGLDWIS GISV--GGASTYYARSVQDVH_106EVQLVQSGGGLVQPGGSLRLSCAASGFNFD D--YPMT WIRQAPGKGLEWVA SIYS--GISTTYYPDSVKGVH3(3-13)EVQLVESGGGLVQPGGSLRLSCAASGFTFS S--YDMH WVRQATGKGLEWVS AIG---TAGDTYYPGSVKGVH_115EVQLVESGGGLVQPGGSLRLSCAASGFTSS T--YAMS WVRQGPGKALEWVS TIN---GADFTSYVDSVKGVH_85EVQLVQSGGGLVQPGGSLRLSCAASGFTFS T--YWMY WVRQAPGKGLEYVG SID---NDGFTYYSEDVKGVH_114EVQLVESGGGLVQPGGSLRLSCTASGFTFS T--HTMS WVRQAPGKGLEWVS GIN---SAYGTIYIDSVKGVH_78EVQLVESGGGLVQPGGSLRLSCAASRFTFG T--SGMT WVRQAPGKGLEWVS TIN---SGGLTTSADSVKG3B5 EVQLVQSGGGLVQPGGSLRLSCAASGFTFS S--YWMY WVRQAPGKGLEWVS TIT---KGGSTYYSDSVKG3A3EVQLVESGGGLVQPGDSLRLSCAASGFTFG N--YDMS WVRQAPGKGPEWVS GIN---SGGKTYSADSVKG              FR3     7         8            967890123456789012abc345678901234 VH1(1-02)RVTMTRDTSISTAYMELSRLRSDDTAVYYCAR 1C2RVTFTADTSTSTAYVELSSLRSEDTAVYYCAR SGRYELDY            WGLGTQVTVSS 1G5RVTFTADTSTSTAYVELSSLRSEDTAVYFCAT SGATMSDLDSFGS       WGQGTQVTVSSVH3(3-23) RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK 5B12RFTISRDNAKNTLYLVMNSLTSEDTAVYYCTK PSTSWSTNYGMDY       WGKGTQVTVSS 1G4RFTISRDSAKNMVHLQMDSLKSEDTAVYYCAK AYRGST              LGQGTQVTVSS 5G7RFTISRDNAKNTLSLQMNSLKPEDTALYYCAR DLRDYYSDYTFVN       WGQGTQVTVSS VH_99RFTISRDNTKGALYLQMNSLKFEDTAVYYCGT RSGTWWRGSYIYTESEENG WGQGTQVTVSS VH_76RFTISRDNAKNTLYLQMNSLKPEDTALYYCAR EGYYSDYAAVGHAYDY    WGQGTQVTVSS VH_86RFTISRDNAKNTLNLQMNSLKPDDAGVYYCAS FGSSAYSWGYLGMDH     WGKGALVTVFS VH_98RFTISRDNAKNTLYLQMNNLKPEDTAVYYCAK GGVLGHSNYYAMDY      WGKGTLVTVSS VH_89RFTIARDKTKNTLVLSMNSLSPEDTAVYYCVA DASALSWSRPALEV      WGQGTLVTVSD VH_82RFTISRDNAKNTLYLQMNSLKAEDKGVYYCGK DESRGIEPGWGSIY      WGQGTQVTVSS VH_68RFTISRDNAKNTLYLQMSSLKPEDTAVYYCTR YDSFGWNVRYGMDY      WGKGTLVTVSS VH_73RLTISRDNAKNTVYLQMNSLKPEDTAVYYCTA ELNWEPENAYSDH       WGQGTQVTVSS VH_107RFTISRDNVKNTLYLQMNSLKPEDTAVYYCTK DPRNSWYTYGMDY       GGKGTLVVVSS VH_94RFTISRDNAKNMVYLQMNSLKPEDTAVYYCAK RIEGGMGYGMDY        WGKGTPVTVSS VH_90RFTISRDNAGNTLYLQMTNLKPEDTAKYFCVK SPEWTYYYGMDS        WGKGTLVTVSS VH_77RFTISRDNAKNTLYLQMNSLKPEDTAVYYCAK SFGLVTGVYFGS        WGQGTQVTVSS VH_74RFTISRDNAKNTMYLQLNSLKPEDTAVYYCAA DSYHAATGYLEQ        WGQGTLVTVSS VH_67RFTISRDNGKDTVYLEMNSLKSDDTGLYYCAR DHRGWGTIRYDY        WGQGTQVTVSS VH_79RFTISRDNAKNTLYLQMNSLKFEDTAVYYCAK GGVIDADHFES         WGQGTQVTVSS VH_75RFTASRDNAKNTLYLTINTLEPNDTAVYYCAK PTSSMWSPGDY         WGQGTQVTVAS VH_110RFTISRDNAKNTLYLEMNDLKPEDTALYYCAR ISTELGNTLDA         WGQGSLVTVSS VH_109RFTITRDNAKNTVYLQMNNLKLDDTAVYYCAK LDVYVDYGMDY         WGKGTLVTVSS VH_103RFTISRDNAQNTLYLQMNSLKPEDTALYYCAR ESGGPGMDLEV         WGQGTLVTVSS VH_84RFTISRDNAKNTLYLQMNSLKPEDTAVYYCAK WEVVTLDFGS          WGQGTQVTVSS VH_113RFTISRDNAKNTLYLQMNSLKPEDMAQYYCVR GGLYGYDYEH          WGQGTQVTVSS VH_87RFTISRDNTVNTVYLQLNSLKPEDTGLYYCAR VGNMWSSDY           WGQGTQVTVSS VH_105RFTISRDNAKNTLYLQMNSLKSEDTALYYCAK ALGYNAFDA           WGRGTLVTVSS VH_101RFTISRDNAKNALYLQMNDLRPDDTAMYYCTN TGPWYTYNY           WGQGTQVTVSS VH_108RFTISRDNTKNTLYLQMSSLRSEDTAVYWCTR GGNTPYDY            WGQGTQVTVSS VH_106RFTISTNDAKNTVYLQMNDLKSDDTAVYYCAN PRRNY               WGQGTHVTVSSVH3(3-13) RFTISRENAKNSLYLQMNSLRAGDTAVYYCAR VH_115RFTISRDNTKNTLYLQMNSLKPEDTAVYYCAK GLSGLNWYGFGDY       WGQGTQVTVSS VH_85RFTISGDNARNTLYLQINSVKPEDTALYYCVR GVYYMDYEPRMDY       WGKGTLVTVSS VH_114RFTISRDNAKNTLYLQMNSLKPDDTAVYYCVQ VVDTWDEYDY          WGQGTQVTVSS VH_78RFTISRDNGKNTLYLQMDSLKPDDTAVYYCAN LLELGH              WGRGTQVTVSS 3B5 RFTISRDNAKNTLYLQMNSLKSEDTAVYYCAK SNSGTHWYEYYGMDY     WGKGTLVTVSS 3A3RFTISRDNAKNTLYLQMNNLKPEDTAVYYCIL GIVTLGS             WGQGTQVTVSS

Example 15 Generating Fabs Against IL-1 Beta

Unless otherwise indicated, the materials and protocols used in thefollowing study were analogous to those used in examples 1-9.

Llamas were Successfully Immunized with IL-1 Beta

Two llamas (Lama glama) were immunized with human IL-1Beta according toa standard protocol (as described in Example 1).

Sera from both llamas were tested for the presence of antibodies againstIL-1Beta by ELISA prior (day 0) and after immunization (day 28). Asshown in FIG. 1, a specific signal against IL-1Beta was observed inELISA after immunization, even after 10,000 fold dilution of the serum.This high antibody titer indicates a specific and appropriate immuneresponse.

Fab Libraries with Good Diversity were Constructed

PBLs isolated from both immunized llamas were used for RNA extraction,RT-PCR and PCR-cloning of Fab in a phagemid using the strategy describedby de Haard et al (JBC 1999), to obtain a diverse library of gooddiversity (2−5×10⁸).

The following primers were used:

Name SequencePrimers for cloning of lambda light chain (SEQ ID NOS: 227-240)HuVI1A-BACK-ApaLI GCC TCC ACC AGT GCA CAGTCTGTGYTGACKCAGCCHuVI1B-BACK-ApaLI GCC TCC ACC AGT GCA CAGTCTGTGYTGACGCAGCCHuVI1C-BACK-ApaLI GCC TCC ACC AGT GCA CAGTCTGTCGTGACGCAGCCHuVI2-BACK-ApaLI GCC TCC ACC AGT GCA CAGTCTGCCCTGACTCAGCCHuVI3A-BACK-ApaLI GCC TCC ACC AGT GCA CTT TCCTATGAGCTGACWCAGCCHuVI3B-BACK-ApaLI GCC TCC ACC AGT GCA CTT TCTTCTGAGCTGACTCAGGAHuVI4-BACK-ApaLI GCC TCC ACC AGT GCA CAGCYTGTGCTGACTCAATCHuVI5-BACK-ApaLI GCC TCC ACC AGT GCA CAGGCTGTGCTGACTCAGCCHuVI6-BACK-ApaLI GCC TCC ACC AGT GCA CTT AATTTTATGCTGACTCAGCCHuVI7/8-BACK-ApaLI  GCC TCC ACC AGT GCA CAGRCTGTGGTGACYCAGGAHuVI9-BACK-ApaLI GCC TCC ACC AGT GCA CWGCCTGTGCTGACTCAGCCHuVI10-BACK-ApaLI GCC TCC ACC AGT GCA CAGGCAGGGCTGACTCAGCCcaClambda1-FOR CTAACACTGGGAGGGGGACACCGTCTTCTC caClambda2-FORCTAACACTGGGAGGGNCTCACNGTCTTCTCPrimers for cloning of kappa light chain (SEQ ID NOS: 241-249)HuVk1B-BACK-ApaLI GCC TCC ACC AGT GCA CTT GACATCCAGWTGACCCAGTCTCCHuVk2-BACK-ApaLI GCC TCC ACC AGT GCA CTT GATGTTGTGATGACTCAGTCTCCHuVk3B-BACK-ApaLI GCC TCC ACC AGT GCA CTT GAAATTGTGWTGACRCAGTCTCCHuVk2/4-BACK-ApaLI GCC TCC ACC AGT GCA CTT GAYATYGTGATGACCCAGWCTCCHuVk5-BACK-ApaLI GCC TCC ACC AGT GCA CTT GAAACGACACTCACGCAGTCTCCHuVk6-BACK-ApaLI GCC TCC ACC AGT GCA CTT GAAATTGTGCTGACTCAGTCTCCHuVk4B-BACK-ApaLI GCC TCC ACC AGT GCA CTT GATATTGTGATGACCCAGACTCCcaCHkapFOR-AscI GCC TCC ACC GGG CGC GCC TTA TTAGCAGTGTCTCCGGTCG AAGCTCCTcaCHkap2FOR-AscI GCC TCC ACC GGG CGC GCC TTA TTARCARTGYCTNCGRTCRAANon-tagged primers for cloning of Heavy chain(step 1) (SEQ ID NOS: 347-351) VH1a-BACK CAGGTKCAGCTGGTGCAGTCTGGVH5a-BACK GARGTGCAGCTGGTGCAGTCTGG VH4a-BACK CAGSTGCAGCTGCAGGAGTCTGGVH4b-BACK CAGGTGCAGCTACAGCAGTCTGG VH2b-BACK CAGGTCACCTTGARGGAGTCTGGTagged primers for cloning of Heavy chain (step 2) (SEQ ID NOS: 352-356)VH1a-BACK-SfiI CTC GCA ACT GCG GCC CAG CCG GCC ATGGCCCAGGTKCAGCTGGTGCAGTCTGG VH5a-BACK-SfiICTC GCA ACT GCG GCC CAG CCG GCC ATG GCCGARGTGCAGCTGGTGCAGTCTGGVH4a-BACK-SfiI CTC GCA ACT GCG GCC CAG CCG GCC ATGGCCCAGSTGCAGCTGCAGGAGTCTGG VH4b-BACK-SfiICTC GCA ACT GCG GCC CAG CCG GCC ATG GCCCAGGTGCAGCTACAGCAGTCTGGVH2b-BACK-SfiI CTC GCA ACT GCG GCC CAG CCG GCC ATGGCCCAGGTCACCTTGARGGAGTCTGG

Independent VλCλ and VκCκ libraries were constructed using a single(tagged)-PCR step (30 cycles) to conserve a greater clonal diversity.

The VHCH1 libraries were built in parallel using a two step PCR (25cycles with non tagged primers (step 1) followed by 10 cycles of taggedprimers (step 2)).

Next, the light chain from the VλCλ and VκCκ libraries are re-clonedseparately in the VHCH1-expressing vector to create the “Lambda” and“Kappa” llama Fab-library respectively (two for each immunized llama).Quality control of the libraries was routinely performed using PCR.

Up to 93% of the clones tested randomly contained full length Fabsequences, indicating a high quality of the libraries.

Human IL-1Beta Specific Fabs were Selected

Phage display was used to identify a large diversity of llama Fabsbinding to biotinylated IL-1Beta. Biotinylated IL-1Beta was used forcapturing to conserve the active conformation of the protein. After tworounds of selection, a good enrichment compared to control was observed.Phage ELISA revealed presence of clones expressing cytokine specificFabs (data not shown).

The phage binding to biotinylated IL-1Beta were eluted by pH shock.Sequential dilutions of the output (10⁻¹ to 10⁻⁵) were used to infectfresh E. coli TG1 cells. The number of colonies obtained indicate thenumber of phage bound during the selection. In the example above, 5 μlof output gave around 10⁵ phage when selection was done with 100 nM and10 nM of biot-IL-1Beta. Compared to the 10² phages obtained bynon-specific binding, this gives a 1000 fold enrichment.

94 Single clones were grown and used to produce monoclonal phage. Thesephage were used in a phage ELISA. Many phage showed good binding tobiot-IL-1Beta after two rounds of selection on biotinylated IL-1Beta.

Human IL-1Beta Specific Fabs have High Starting Homology to HumanGermline

Target specific VH and Vλ domains were matched with those common humangermlines showing an identical CDR1 and CDR2 length and correspondingcanonical folds. Subsequently the closest human germline was selectedbased on sequence homology in their framework regions. Non-matchingamino acid residues were checked for their presence in other, relatedhuman germlines. In case there was no match, these residues were countedas foreign.

TABLE 7 Overall sequence homology of llama VH to human germline ClosestHuman % Sequence Germline Matching Clones Homology IGHV1-2 1C2/2B7/2C1293 2D8 94 1G5/2D7 93 2E12/2G7 94 IGHV3-23 1F2 98 1G4 92 5G7 95 5B12 921A1/2B8/2B9 98 1C3/1E3/2A7 98 IGHV3-13 1E2 94 3A3/3B6/3E2/3E3 95 3B5/4F198 IGHV3-20 4H1 94 4H4 93

TABLE 8 Overall sequence homology of llama VL to human germline ClosestHuman Matching % Sequence Germline Clones Homology IGLV8-61 1E2 90 1F290 3E2/3E3/3A3 86 3B5/4F1 86 IGLV2-18 3B6 91 IGLV5-52 1G4 VL 96 IGLV3-191C2/2B7/2D7 95 2E12/2G7 96 2D8 95

Discussion and Conclusions:

-   -   A total of 14 target specific VH families, 9 target specific Vλ        families and 3 Vκ families were identified based on this very        first selection    -   This initial panel of 14 anti-IL-1Beta WT VH's and 12        anti-IL-1Beta WT VL's shows a remarkably high sequence homology        to the human germline.    -   33% of those VH domains have a starting homology of 95% or more        to the human situation and about 44% of the VL domains have a        starting homology of 95% or more to the human situation,        eliminating the need for further humanization.    -   VH domain 2D8 is a humanized version of VH1C2 because it has one        deviating amino acid residue less as compared to the closest        human germline. Its corresponding VL domain (VL 2D8), had a        starting homology of 95% which was further increased to 96% by 1        back mutation (VL 2G7) to the closest human germline.    -   All VH and VL domains, without a single exception, exhibited        human 3-D binding site structures (i.e. identical combinations        of canonical folds for CDR1 and CDR2 as occurring in the        matching human germline segments) when assessed using the        methodology described above (data not shown).

Humanization of Fabs 1E2 and 1F2

Humanization was performed on two IL-1Beta specific Fabs coded 1E2 and1F2. Based on the alignment against the closest human germlines,mutations in their VH and Vλ framework regions were proposed (FIG. 2).The germlining of VH matching to the human VH3 family will often involvea number of residues, which already deviate in publically known Lamaglama, Lama pacos or Camelus dromedarius derived germline sequences. Forinstance, Alanine on position 71 (numbering according to Kabat) andLysine on 83 and Proline on 84 might be changed into Serine (althoughAlanine exists in certain human germline VH3 members), Arginine(although Lysine is used by a number of human VH3 germlines) andAlanine, respectively. For light chain variable sequences no germlinesequences are available for Camelids, but it is very likely that anumber of deviations in FRs from human germline exists that will bechanged in the majority of lead antibodies. Besides the fully humanized(hum) and the wild type (wt) V regions, also a “safe variant” with onlythree wild type residues remaining was proposed (safe).

Fab 1E2 was formatted in a step-by-step approach, whereby the differentversions (wt, safe and fully humanized) of the Vλ fused to the humanconstant domain were combined with various versions of the VH fused tothe human constant CH1 domain to generate the Fabs indicated in Table 9.

TABLE 9 Fab 1E2 formats VH 1E2 + hum CH1 wt safe hum VL 1E2 + humCL wtwt 1E2 wt/safe wt/hum safe safe/wt safe 1E2 safe/hum hum hum/wt hum/safehum 1E2

The genes of these Fabs were ordered as synthetic genes with GeneArt(Germany) and were subsequently produced in E. coli, purified and testedfor their ability to bind biot-IL-1Beta. For this the Fabs were capturedon an anti-myc coated Maxisorp plate. Biotinylated human IL-1Beta wasadded and bound cytokine was detected using HRP-conjugated streptavidin.The read out of this assay is represented in FIG. 3 below.

-   -   The replacement of the wild type constant domains CH1 and Cλ by        their human counterpart did not affect the binding capacity.    -   Partial (wt Vλ/safe VH) and complete (wt Vλ/hum VH) humanization        of the VH domain of 1E2 generated a functional Fab.

The humanized variants of clone 1F2 were tested with phage expressinggene3-Fabs fusions (FIG. 4). Phage were produced from 4 independentclones for each construct:

-   -   wt 1F2 and wt 1E2 (llama Vλ and VH fused to human Cλ and CH1)    -   safe variant 1F2 and safe variant 1E2 (partially humanized Vλ        fused to human Cλ and CH1)    -   hum 1F2 and hum 1E2 (fully humanized Vλ and VH fused to human Cλ        and CH1)

Four clones for each Fab were tested to overcome clonal variation (dueto bacterial growth, phage production efficiency and toxicity etc. . . .). Phage ELISA was performed by capturing of biotinylated IL-1Beta onneutravidin coated Maxisorp plate and subsequent incubation of crudephage extract (i.e. bacterial medium). After extensive washing, boundphages were detected with an anti-M13-HRP monoclonal antibody. The samephage preparations when tested on neutravidin coated wells (withoutbiotinylated IL-1Beta) did not give signals (data not shown).

-   -   Back mutations in the framework regions of 1F2 VL and VH domains        to the closest human germline successfully yield partially        (safe) and fully (hum) humanized variants, maintaining antigen        specificity.

Successful Formatting of Camelid Variable Domains with Human ConstantDomains

The VL and VH variable domains of the IL-1Beta specific clone 1E2 weresuccessfully fused to the human Cλ and CH1 constant domains, resultingin a “chimeric” Fab which was produced and purified.

This chimeric 1E2 Fab was produced by performing the induction for 4 hat 37° C. (o/d) or for 16 h and 28° C. (o/n) from the pCB5 phagemid(Δgene3). The wild type llama 1E2 Fab was produced by performinginduction for 16 h at 30° C. from pCB3 (gene3 containing phagemid).After purification, these Fabs were loaded on SDS-PAGE with (reducing)or without (non-reducing) DTT. Coomassie staining was performed, nicelyshowing the presence of these Fabs or their composing light and heavychains at the expected molecular weight band (not shown).

The purified llama and chimeric 1E2 Fabs described above were capturedon anti-myc coated maxisorp plates. After incubation with biotinylatedhuman IL-1Beta and extensive washing, the biotinylated IL-1Beta bound tothe Fab was detected using HRP-conjugated streptavidin. Both thepurified llama and chimeric 1E2 Fabs exhibited functional target binding(FIG. 5). This finding demonstrates the feasibility to associate Camelidderived variable domains with the constant domains of human IgGs

A Subset of Fabs Showed Functional Inhibition of the Target

The table below shows the OD values resulting from the following ELISAexperiment. Wells were coated with a mouse monoclonal antibody known toinhibit the binding of IL1-Beta with its receptor (provided by DiacloneSAS).

Biotinylated IL-1Beta was added to the wells together with periplasmicextracts of Fabs identified after 2 rounds of selection againstbiotinylated IL-1Beta. Detection of the bound biotinylated IL-1Betahappened through HRP labeled streptavidin. A reduced signal indicatedthe competition of a specific Fab with the blocking mouse monoclonalantibody, suggesting antagonism.

A positive control was included in well G12 by spiking in a large amountof the competing mouse monoclonal (well 12G in Table 10).

TABLE 10 Results of competition assay

A number of Fabs were identified which successfully compete with theblocking mouse monoclonal antibody (indicated by shaded cells in Table10). Sequence analysis of the competing clones revealed the presence ofthree Fabs with different VH, which were present in 48 screened clones(part of the plate coded 6A in Table 10). The sequences alignments (SEQID NO: 21 and SEQ ID NO: 250; SEQ ID NO: 21 and SEQ ID NO: 251; and SEQID NOS: 254, 252, and 253) against the closest human germline and thestructural homology analysis of the VH of the antagonistic Fab 1A1(giving a signal of 0.205 in the competition assay of Table 10), 1B3(signal of 0.444) and the related clone 1G1 (signal of 0.498) andfinally 1C3 (signal of 0.386) are shown below.

All three have a very high degree of sequence homology with the matchinghuman germline and have the identical canonical fold combinations asfound in the human germline. This was also observed for the lambda lightchain in the three antagonistic leads (data not shown). Fab 1A1 competesstrongly with the antagonistic reference monoclonal antibody (IC50 of 12μg/ml in ELISA based competition assay), whereas Fab 1C3 hardly showscompetition (only at concentrations of more than 50 μg/ml). However, inthe bioassay 1C3 is more potent (IC50 of 3 μg/ml) than 1A1 (IC50 of 10μg/ml), which suggests a different epitope recognition. The highfrequency of different antagonistic Fabs (3 different antibodies in 48screened clones) and the difference in epitope recognition as found fortwo of these illustrates the high diversity of antibodies as the resultof the outbred nature of the lama. The high degree of sequence homologywith human germline V regions combined with the high diversity of(potent) antibodies and the broad epitope coverage enables theidentification of panels of therapeutic antibodies from immunizedcamelids.

IGHV gene CDR1 CDR2 Kabat FR/CDR FR1-Kabat Kabat FR2-Kabat KabatIMGT numbering   M99660,IGHV3-23 VH-IA1,3B8,3D9

IGHV gene CDR3 Kabat FR/CDR FR3-Kabat Kabat FR4 (IGHJ) IMGT numbering  M99660,IGHV3-23 VH-IA1,3B8,3D9

5V primer encoded and also in all other class 3 except 3-23 83Aoccurs in all VH3 family members except 3-23 95 Kalso in IGHV3-13/49/66/72 and classes 5 and 7 96 Snot in class 3 but in class 1 127 Q not in human germlineOverall homology 84/86 framework residues = 98% homologyCanonical folds analysis CDR H1 Class 1/10A [2fbj] CDR H2 Class ? !Similar to class 3/10B, but: ! H56 (Chothia Numbering) =A (allows: SYTNDR) IGHV gene CDR1 CDR2 Kabat FR/CDR FR1-Kabat KabatFR2-Kabat Kabat IMGT numbering   M99660,IGHV3-23 VH-1C3,1E3,2A7

IGHV gene CDR3 Kabat FR/CDR FR3-Kabat Kabat FR4 (IGHJ) IMGT numbering  M99660,IGHV3-23 VH-1C3,1E3,2A7

5V primer encoded and also in all other class 3 except 3-23 83Aoccurs in all VH3 family members except 3-23 95 Kalso in IGHV3-13/49/66/72 and classes 5 and 7 96 Snot in class 3 but in class 1 127 Q not in human germlineOverall homology 84/86 framework residues = 98% homologyCanonical folds analysis CDR H1 Class 1/10A [2fbj] CDR H2 Class ? !Similar to class 2/10A, but: ! H71 (Chothia Numbering) = R (allows: VAL)

Example 16

The following example demonstrates the functional diversity which can beachieved with the current invention, in comparison with the establishedmouse monoclonal antibody approach.

10 BALB/c mice were immunized with a recombinant produced cytokine witha small molecular weight. After completion of the immunization protocol,the animals were sacrificed, and hybridomas were generated byimmortalizing their spleen cells. Supernatant of the resultinghybridomas was tested in the cytokine binding ELISA and subsequently ina suitable bioassay. One highly potent antagonist and one weakerantagonist could be identified.

Also, 4 llamas were immunized with the same recombinant producedcytokine, using the general protocol described herein. After completionof the immunization protocol, peripheral B lymphocytes were harvestedand their RNA was extracted and purified. Using a set of llama specificprimers, Fab libraries were generated using the phage display technique.Those Fabs were tested in the cytokine/cytokine receptor binding ELISA.5 different VH families could be identified from the first 2 llamas, and6 additional different VH families from the next 2 llamas, which blockedthe cytokine/receptor interaction with high potency, meaning that thoseVH domains contained uniquely different CDRs, both in length and aminoacid sequence.

Thus a higher functional diversity could be achieved from a small numberof outbred llamas as opposed to a higher number of inbred BALB/c mice.All VH families obtained by active immunisation of llamas exhibited anextraordinary sequence homology as compared to the closest humangermline and had the same canonical fold combinations for CDR1 and CDR2as the matching human germlines.

Example 17

The following table summarises the results of amino acid sequencehomology comparisons between germline VH domains of alpaca (Lama pacos)and the closest matching human germline VH domains. % homology wascalculated using the same algorithm as described herein for Lama glama.Raw VH sequence data for Lama pacos is not shown:

TABLE 11 amino acid sequence homology germline VH of Lama pacos vs human% amino acid sequence homology with closest Alpaca (Lama pacos) matchinghuman germline germline VH family Frequency VH VH3 70% (36/51) ≧95% VH110% (5/51)  90-92% VH2 (NB Lama pacos VH2 20% (10/51) 81-88% aligns tohuman VH4)

The following table is provided to cross-reference nucleotide and aminoacid sequences listed herein with the sequence listing submitted inST.25 format for searching purposes.

TABLE 12 cross-reference to sequence identifiers SEQ ID No. Sequencename 1 M99679, IGHV3-53 2 AF000603, IGHV1S1 3 AJ245151, IGHV1S2 4AJ245152, IGHV1S3 5 AJ245153, IGHV1S4 6 AJ245154, IGHV1S5 7 AJ245155,IGHV1S6 8 AJ245157, IGHV1S7 9 AJ245158, IGHV1S8 10 AJ245159, IGHV1S9 11AJ245160, IGHV1S10 12 AJ245164, IGHV1S11 13 AJ245165, IGHV1S12 14AJ245167, IGHV1S13 15 AJ245168, IGHV1S14 16 AJ245170, IGHV1S15 17AJ245171, IGHV1S16 18 AJ245173, IGHV1S17 19 AJ245174, IGHV1S18 20AJ245156, IGHV1S19 21 M99660, IGHV3-23 22 AJ245177, IGHV1S20 23AJ245178, IGHV1S21 24 AJ245183, IGHV1S22 25 AJ245185, IGHV1S23 26AJ245186, IGHV1S24 27 AJ245187, IGHV1S25 28 AJ245189, IGHV1S26 29AJ245191, IGHV1S27 30 AJ245192, IGHV1S28 31 AJ245193, IGHV1S29 32AJ245194, IGHV1S30 33 AJ245195, IGHV1S31 34 AJ245179, IGHV1S32 35AJ245180, IGHV1S33 36 AJ245182, IGHV1S34 37 AJ245190, IGHV1S35 38AJ245196, IGHV1S36 39 AJ245197, IGHV1S37 40 AJ245181, IGHV1S38 41AJ245198, IGHV1S39 42 AJ245199, IGHV1S40P 43 AF305949, IGHV1S6 44M94116, IGLV1-40 45 Camv144 46 Z73642, IGLV2-18 47 Camvl17 48 Camvl33 49Camvl36 50 Camvl59 51 Camvl30 52 Camvl32 53 Camvl57 54 Camvl5 55 Camvl6556 Camvl51 57 Camvl31 58 Camvl60 59 Camvl52 60 X57826, IGLV3-1 61Camvl19 62 Camvl20 63 Camvl8 64 Camvl18 65 Camv123 66 Z73658, IGLV3-1267 Camvlll 68 X59314, IGKV2-40 69 Kp6 70 Kp48 71 Kp3 72 Kp20 73 Kp7 74Kp10 75 Kp1 76 J00256, IGHJ1 77 J00256, IGHJ2 78 J00256, IGHJ3 79J00256, IGHJ4 80 J00256, IGHJ5 81 J00256, IGHJ6 82 AF305952, IGHJ2 83AF305952, IGHJ3 84 AF305952, IGHJ4 85 AF305952, IGHJ5 86 AF305952, IGHJ6(1) 87 X04457, IGLJ1 88 M15641, IGLJ2 89 M15642, IGLJ3 90 X51755, IGLJ491 X51755, IGLJ5 92 M18338, IGLJ6 93 X51755, IGLJ7 94 Camvl19 etc. 95Camvl8/18/4 96 Camvl58/28/5 97 J00242, IGKJ1 98 J00242, IGKJ2 99 J00242,IGKJ3 100 J00242, IGKJ4 101 J00242, IGKJ5 102 Kp6/48/3/20/10/1 103 Kp7(1/7 analyzed) 104 M99660, IGHV3-23 105 IVH28 106 IVH69 107 IVH47 108IVH48 109 IVH70 110 IVH71 111 HuVL1-1(3/4/5) 112 LAMBDA#14 113 LAMBDA#16114 LAMBDA#46 115 LAMBDA#45 116 LAMBDA#15 117 HuVL1-2 118 LAMBDA#47 119LAMBDA#18 120 LAMBDA#32 121 LAMBDA#28 122 LAMBDA#29 123 LAMBDA#27 124LAMBDA#17 125 LAMBDA#4 126 LAMBDA#7 127 LAMBDA#8 128 LAMBDA#5 129HuVL2-1 130 LAMBDA#1 131 LAMBDA#8 (2) 132 LAMBDA#5 (2) 133 LAMBDA#11 134HuVL3-2 135 LAMBDA#13 136 LAMBDA#40 137 LAMBDA#2 138 LAMBDA#14 (2) 139HuVL4-1 140 LAMBDA#10 141 HuVL5-1 142 LAMBDA#3 143 LAMBDA#7 (2) 144LAMBDA#9 145 LAMBDA#26 146 LAMBDA#12 147 HuVL8-1 148 LAMBDA#42 149LAMBDA#31 150 HuVK1-1 151 KAPPA#39 152 KAPPA#22 153 KAPPA#24 154KAPPA#21 155 KAPPA#23 156 KAPPA#7 157 KAPPA#19 158 KAPPA#25b 159 I-_8160 KAPPA#43 161 KAPPA#44 162 KAPPA#20b 163 HuVK4-1 164 KAPPA#53 165KAPPA#50 166 KAPPA#20 167 KAPPA#48 168 KAPPA#55 169 KAPPA#9 170 KAPPA#51171 KAPPA#10 172 KAPPA#54 173 KAPPA#49 174 KAPPA#52 175 KAPPA#13 176KAPPA#11 177 KAPPA#36 178 KAPPA#25 179 KAPPA#12 180 KAPPA#34 181KAPPA#48b 182 KAPPA#21 (2) 183 KAPPA#24 (2) 184 KAPPA#22b 185 KAPPA#45186 KAPPA#46 187 VH1(1-02) 188 1C2 189 1G5 190 VH3(3-23) 191 5B12 1921G4 193 5G7 194 VH_99 195 VH_76 196 VH_86 197 VH_98 198 VH_89 199 VH_82200 VH_68 201 VH_73 202 VH_107 203 VH_94 204 VH_90 205 VH_77 206 VH_74207 VH_67 208 VH_79 209 VH_75 210 VH_110 211 VH_109 212 VH_103 213 VH_84214 VH_113 215 VH_87 216 VH_105 217 VH_101 218 VH_108 219 VH_106 220VH3(3-13) 221 VH_115 222 VH_85 223 VH_114 224 VH_78 225 3B5 226 3A3 227HuVl1A-BACK-ApaLI 228 HuVl1B-BACK-ApaLI 229 HuVl1C-BACK-ApaLI 230HuVl2-BACK-ApaLI 231 HuVl3A-BACK-ApaLI 232 HuVl3B-BACK-ApaLI 233HuVl4-BACK-ApaLI 234 HuVl5-BACK-ApaLI 235 HuVl6-BACK-ApaLI 236HuVl7/8-BACK-ApaLI 237 HuVl9-BACK-ApaLI 238 HuVl10-BACK-ApaLI 239caClambda1-FOR 240 caClambda2-FOR 241 HuVk1B-BACK-ApaLI 242HuVk2-BACK-ApaLI 243 HuVk3B-BACK-ApaLI 244 HuVk2/4-BACK-ApaLI 245HuVk5-BACK-ApaLI 246 HuVk6-BACK-ApaLI 247 HuVk4B-BACK-ApaLI 248caCHkapFOR-AscI 249 caCHkap2FOR-AscI 250 VH-1A1, 2B8, 2B9 251 VH-1C3,1E3, 2A7 252 VH-1B3, 1G2, 2D8 253 VH-1G1, 2B7 254 X07448, IGHV1-2 2551F2 VH 256 Humanized VH 1F2 257 Safe Variant VH 1F2 258 X92218, IGHV3-66259 1E2 VH 260 Humanized VH 1E2 261 Safe Variant VH 1E2 262 Z73650,IGLV8-61 263 1F2 VL 264 Humanized 1F2 VL 265 Safe Variant VL 1F2 266 1E2VL 267 Humanized VL 1E2 268 Safe Variant VL 1E2 269 X92343|IGHV1-46*01270 LpVH1-s6 (AM939701) 271 LpVH1-s2 (AM939697) 272 LpVH1-s3 (AM939698)273 LpVH1-s4 (AM939699) 274 LpVH1-s5 Ps (AM939700) 275M99660|IGHV3-23*01 276 AM939712 277 AM939713 278 AM939730 279 AM939731280 AM939744 281 AM939726 282 AM939727 283 AM939739 284 AM939740 285AM939741 286 AM939742 287 AM939743 288 U29481|IGHV3-23*03 289 AM939716290 AM939728 291 AM939738 292 AM939710 293 AM939748 294 Am939750 295AM939751 296 AM939767 297 AM939768 298 AM939707 299 AM939708 300AM939709 301 AM939732 302 AM939733 303 AM939717 304 AM939734 305AM939735 306 AM939736 307 AM939737 308 L33851|IGHV3-74*01 309 AM939749310 AM939724 311 AM939725 312 AM939745 313 AM939723 314X92229|IGHV4-30-2*03 315 LpVH2-s7 (AM939704) 316 Z14238|IGHV4-30-4*01317 LpVH2-s2 (AM939769) 318 LpVH2-s3 (AM939770) 319 LpVH2-s4 (AM939771)320 LpVH2-s5 (AM939772) 321 LpVH2-s6 (AM939773) 322 LpVH2-s11 Ps(AM939703) 323 LpVH2-s8 (AM939705) 324 LpVH2-s9 Ps (AM939706) 325LpVH2-s10 (AM939702) 326 AJ879486|IGHV3-23*04 327 S-VH1 328 S-VH3 329S-VH4 330 S-VH2 331 S-VH6 332 S-VH5 333 D86994|IGLV3-25*02 334 VL25-28335 Z73672|IGLV5-37*01 336 VL2, 12, 15 337 Z73650|IGLV8-61*01 338 VL3, 5339 VL17-24, 29-32 340 VL10 341 VL4, 6, 7, 8, 9, 13, 14 342U41644|IGKV2D-29*02 343 KAPPA 33-36, 38, 39, 42, 4 344 KAPPA 41, 43, 44345 KAPPA 40, 44 346 KAPPA 37, 46, 48 347 VH1a-BACK 348 VH5a-BACK 349VH4a-BACK 350 VH4b-BACK 351 VH2b-BACK 352 VH1a-BACK-SfiI 353VH5a-BACK-SfiI 354 VH4a-BACK-SfiI 355 VH4b-BACK-SfiI 356 VH2b-BACK-SfiI

The invention claimed is:
 1. A chimeric antigen binding polypeptide thatspecifically binds to a target antigen, the polypeptide comprising a VHdomain and a VL domain, said VH domain comprising hypervariable loopsH1, H2 and H3, wherein said VH domain polypeptide is paired with a VLdomain comprising hypervariable loops L1, L2 and L3, wherein said VHdomain and said VL domain are each fused to one or more IgG constantdomains of a human antibody, wherein at least the H3 hypervariable loopis obtained from a conventional antibody of a species in the familyCamelidae, and wherein said conventional antibody is a heterotetramericIgG antibody composed of two identical light chains and two identicalheavy chains, wherein at least one of the hypervariable loops H1, H2,L1, L2 and L3 exhibits a predicted or actual canonical fold structurewhich is identical or substantially identical to a canonical foldstructure of a corresponding H1, H2, L1, L2 or L3 hypervariable loopwhich occurs in human antibodies.
 2. The chimeric antigen bindingpolypeptide of claim 1 wherein either hypervariable loop H1 orhypervariable loop H2, or both hypervariable loop H1 and hypervariableloop H2 are obtained from the conventional antibody of a species in thefamily Camelidae.
 3. The chimeric antigen binding polypeptide of claim 1wherein either hypervariable loop L1 or hypervariable loop L2 or bothhypervariable loop L1 and hypervariable loop L2 are obtained from theconventional antibody of a species in the family Camelidae.
 4. Thechimeric antigen binding polypeptide of claim 1 wherein hypervariableloop L3 is obtained from the conventional antibody of a species in thefamily Camelidae.
 5. The chimeric antigen binding polypeptide of claim 1wherein each of the hypervariable loops H1-H3 and L1-L3 are obtainedfrom the conventional antibody of a species in the family Camelidae. 6.The chimeric antigen binding polypeptide of claim 1 wherein theconventional antibody is obtained by active immunisation of the speciesin the family Camelidae with the target antigen, or with apolynucleotide encoding said target antigen.
 7. The chimeric antigenbinding polypeptide of claim 6 which contains at least one amino acidsubstitution in at least one hypervariable loop in comparison to acorresponding hypervariable loop in the conventional antibody.
 8. Thechimeric antigen binding polypeptide of claim 6 which contains at leastone amino acid substitution in at least one framework region of eitherthe VH domain or the VL domain, in comparison to a correspondingframework region in the conventional antibody.
 9. The chimeric antigenbinding polypeptide of claim 1 wherein the VH domain of the polypeptideexhibits a sequence identity of 90% or greater with one or more human VHdomains across the framework regions FR1, FR2, FR3 and FR4.
 10. Thechimeric antigen binding polypeptide of claim 9 wherein the VH domain ofthe polypeptide exhibits a sequence identity of 95% or greater with oneor more human VH domains across the framework regions FR1, FR2, FR3 andFR4.
 11. The chimeric antigen binding polypeptide of claim 10 whereinthe VH domain of the polypeptide exhibits a sequence identity of 97% orgreater with one or more human VH domains across the framework regionsFR1, FR2, FR3 and FR4.
 12. The chimeric antigen binding polypeptide ofclaim 1 wherein the VL domain of the polypeptide exhibits a sequenceidentity of 90% or greater with one or more human VL domains across theframework regions FR1, FR2, FR3 and FR4.
 13. The chimeric antigenbinding polypeptide of claim 12 wherein the VL domain of the polypeptideexhibits a sequence identity of 95% or greater with one or more human VLdomains across the framework regions FR1, FR2, FR3 and FR4.
 14. Thechimeric antigen binding polypeptide of claim 13 wherein the VL domainof the polypeptide exhibits a sequence identity of 97% or greater withone or more human VL domains across the framework regions FR1, FR2, FR3and FR4.
 15. The chimeric antigen binding polypeptide of claim 1 whereinhypervariable loop H1 and hypervariable loop H2 each exhibit a predictedor actual canonical fold structure which is identical or substantiallyidentical to a canonical fold structure of a corresponding H1 or H2hypervariable loop which occurs in human antibodies.
 16. The chimericantigen binding polypeptide of claim 15 wherein hypervariable loop L1and hypervariable loop L2 each exhibit a predicted or actual canonicalfold structure which is substantially identical or substantiallyidentical to a canonical fold structure of a corresponding L1 or L2hypervariable loop which occurs in human antibodies.
 17. The chimericantigen binding polypeptide of claim 16 wherein hypervariable loop L1and hypervariable loop L2 in the VL domain form a combination ofpredicted or actual canonical fold structures which is identical orsubstantially identical to a combination of canonical fold structuresknown to occur in corresponding hypervariable loops in a human germlineVL domain.
 18. The chimeric antigen binding polypeptide of claim 17wherein the combination of canonical fold structures occurring in thehuman VL domain is selected from the group consisting of: 11-7,13-7(A,B,C), 14-7(A,B), 12-11, 14-11, 12-12, 2-1, 3-1, 4-1 and 6-1. 19.The chimeric antigen binding polypeptide of claim 1 whereinhypervariable loop H1 and hypervariable loop H2 form a combination ofpredicted or actual canonical fold structures which is identical orsubstantially identical to a combination of canonical fold structuresknown to occur in corresponding hypervariable loops in a human germlineVH domain.
 20. The chimeric antigen binding polypeptide of claim 19wherein the combination of canonical fold structures occurring in thehuman VH domain is selected from the group consisting of: 1-1, 1-2, 1-3,1-4, 1-6, 2-1, 3-1 and 3-5.
 21. The chimeric antigen binding polypeptideof claim 1 wherein the target antigen is a non-camelid antigen.
 22. Thechimeric antigen binding polypeptide of claim 21 wherein the targetantigen is a human antigen.
 23. The chimeric antigen binding polypeptideof claim 21 wherein the target antigen is a viral antigen or a bacterialantigen.
 24. The chimeric antigen binding polypeptide of claim 1 whereinthe target antigen is a target of therapeutic importance.
 25. Thechimeric antigen binding polypeptide of claim 1 which is an Fab, Fab',F(ab′)2, bi-specific Fab′, Fv fragments, diabody, linear antibody, asingle chain variable fragment (scFv), antibody or multispecificantibody formed from antibody fragments.
 26. The chimeric antigenbinding polypeptide of claim 1, wherein the species in the familyCamelidae is camel, llama, dromedary, vicunia, guanaco or alpaca. 27.The chimeric antigen binding polypeptide of claim 1, which is anantibody.
 28. The chimeric antigen binding polypeptide of claim 1,wherein said IgG constant domains comprise at least one of a CH1 domain,hinge region, CH2 domain, and CH3 domain of the human antibody.
 29. Thechimeric antigen binding polypeptide of claim 28, wherein the IgGconstant domains further comprise a human CL domain.
 30. Apharmaceutical formulation comprising the chimeric antigen bindingpolypeptide of claim 1 and at least one pharmaceutically acceptablediluent, excipient or carrier.
 31. A test kit comprising the chimericantigen binding polypeptide of claim
 1. 32. The test kit of claim 31which additionally comprises at least one additional reagent required toperform an immunoassay using said chimeric antigen binding polypeptide.